TRANSPORT OF PROTEINS AND NUCLEIC ACIDS THROUGH PLASMODESMATA

Annual Review of Plant Physiology and Plant Molecular Biology. Jun, 1997  |  Pubmed ID: 15012255

Despite a potentially key role in cell-to-cell communication, plant intercellular connections-the plasmodesmata-have long been a biological "black box." Little is known about their protein composition, regulatory mechanisms, or transport pathways. However, recent studies have shed some light on plasmodesmal function. These connections have been shown to actively traffic proteins and protein-nucleic acid complexes between plant cells. This review describes these transport processes-specifically, cell-to-cell movement of plant viruses as well as endogenous cellular proteins-and discusses their possible mechanism(s). For comparison and to provide a broader perspective on the plasmodesmal transport process, the current model for nuclear import, the only other known example of transport of large proteins and protein-nucleic acid complexes through a membrane pore, is summarized. Finally, the function of plasmodesmata as communication boundaries within plant tissue is discussed.

Partners-in-infection: Host Proteins Involved in the Transformation of Plant Cells by Agrobacterium

Trends in Cell Biology. Mar, 2002  |  Pubmed ID: 11859024

Genetic modification of plant cells by Agrobacterium is the only known natural example of DNA transport between kingdoms. While the bacterial factors involved in Agrobacterium infection have been relatively well characterized, studies of their host cellular partners are just beginning. Here, we describe the plant cell factors that might participate in Agrobacterium-mediated genetic transformation and discuss their possible roles in this process. Because Agrobacterium probably adapts existing cellular processes for its life cycle, identifying the host factors participating in Agrobacterium infection might contribute to a better understanding of such basic biological processes as cell communication, intracellular transport and DNA repair and recombination as well as help expand the host range of Agrobacterium as a genetic engineering tool.

The Systemic Movement of a Tobamovirus is Inhibited by a Cadmium-ion-induced Glycine-rich Protein

Nature Cell Biology. Jul, 2002  |  Pubmed ID: 12055637

Systemic movement is central to plant viral infection. Exposure of tobacco plants to low levels of cadmium ions blocks the systemic spread of turnip vein-clearing tobamovirus (TVCV). We identified a tobacco glycine-rich protein, cdiGRP, specifically induced by low concentrations of cadmium and expressed in the cell walls of plant vascular tissues. Constitutive cdiGRP expression inhibited systemic transport of TVCV, whereas suppression of cdiGRP production allowed TVCV movement in the presence of cadmium. cdiGRP exerted its inhibitory effect on TVCV transport by enhancing callose deposits in the vasculature. So cdiGRP may function to control plant viral systemic movement.

Increasing Plant Susceptibility to Agrobacterium Infection by Overexpression of the Arabidopsis Nuclear Protein VIP1

Proceedings of the National Academy of Sciences of the United States of America. Aug, 2002  |  Pubmed ID: 12124400

Agrobacterium is a unique model system as well as a major biotechnological tool for genetic manipulation of plant cells. It is still unknown, however, whether host cellular factors exist that are limiting for infection, and whether their overexpression in plant cells can increase the efficiency of the infection. Here, we examined the effect of overexpression in tobacco plants of an Arabidopsis gene, VIP1, which encodes a recently discovered cellular protein required for Agrobacterium infection. Our results indicate that VIP1 is imported into the plant cell nucleus via the karyopherin alphadependent pathway and that elevated intracellular levels of VIP1 render the host plants significantly more susceptible to transient and stable genetic transformation by Agrobacterium, probably because of the increased nuclear import of the transferred-DNA.

Identification of Arabidopsis Rat Mutants

Plant Physiology. Jun, 2003  |  Pubmed ID: 12805582

Limited knowledge currently exists regarding the roles of plant genes and proteins in the Agrobacterium tumefaciens-mediated transformation process. To understand the host contribution to transformation, we carried out root-based transformation assays to identify Arabidopsis mutants that are resistant to Agrobacterium transformation (rat mutants). To date, we have identified 126 rat mutants by screening libraries of T-DNA insertion mutants and by using various "reverse genetic" approaches. These mutants disrupt expression of genes of numerous categories, including chromatin structural and remodeling genes, and genes encoding proteins implicated in nuclear targeting, cell wall structure and metabolism, cytoskeleton structure and function, and signal transduction. Here, we present an update on the identification and characterization of these rat mutants.

Modes of Intercellular Transcription Factor Movement in the Arabidopsis Apex

Development (Cambridge, England). Aug, 2003  |  Pubmed ID: 12835390

A recent and intriguing discovery in plant biology has been that some transcription factors can move between cells. In Arabidopsis thaliana, the floral identity protein LEAFY has strong non-autonomous effects when expressed in the epidermis, mediated by its movement into underlying tissue layers. By contrast, a structurally unrelated floral identity protein, APETALA1, has only limited non-autonomous effects. Using GFP fusions to monitor protein movement in the shoot apical meristem and in floral primordia of Arabidopsis, we found a strong correlation between cytoplasmic localization of proteins and their ability to move to adjacent cells. The graded distribution of several GFP fusions with their highest levels in the cells where they are produced is compatible with the notion that this movement is driven by diffusion. We also present evidence that protein movement is more restricted laterally within layers than it is from L1 into underlying layers of the Arabidopsis apex. Based on these observations, we propose that intercellular movement of transcription factors can occur in a non-targeted fashion as a result of simple diffusion. This hypothesis raises the possibility that diffusion is the default state for many macromolecules in the Arabidopsis apex, unless they are specifically retained.

Systemic Movement of a Tobamovirus Requires Host Cell Pectin Methylesterase

The Plant Journal : for Cell and Molecular Biology. Aug, 2003  |  Pubmed ID: 12887589

Systemic movement of plant viruses through the host vasculature, one of the central events of the infection process, is essential for maximal viral accumulation and development of disease symptoms. The host plant proteins involved in this transport, however, remain unknown. Here, we examined whether or not pectin methylesterase (PME), one of the few cellular proteins known to be involved in local, cell-to-cell movement of tobacco mosaic virus (TMV), is also required for the systemic spread of viral infection through the plant vascular system. In a reverse genetics approach, PME levels were reduced in tobacco plants using antisense suppression. The resulting PME antisense plants displayed a significant degree of PME suppression in their vascular tissues but retained the wild-type pattern of phloem loading and unloading of a fluorescent solute. Systemic transport of TMV in these plants, however, was substantially delayed as compared to the wild-type tobacco, suggesting a role for PME in TMV systemic infection. Our analysis of virus distribution in the PME antisense plants suggested that TMV systemic movement may be a polar process in which the virions enter and exit the vascular system by two different mechanisms, and it is the viral exit out of the vascular system that involves PME.

The Agrobacterium-plant Cell Interaction. Taking Biology Lessons from a Bug

Plant Physiology. Nov, 2003  |  Pubmed ID: 14612580

Site-specific Integration of Agrobacterium Tumefaciens T-DNA Via Double-stranded Intermediates

Plant Physiology. Nov, 2003  |  Pubmed ID: 14551323

Agrobacterium tumefaciens-mediated genetic transformation involves transfer of a single-stranded T-DNA molecule (T strand) into the host cell, followed by its integration into the plant genome. The molecular mechanism of T-DNA integration, the culmination point of the entire transformation process, remains largely obscure. Here, we studied the roles of double-stranded breaks (DSBs) and double-stranded T-DNA intermediates in the integration process. We produced transgenic tobacco (Nicotiana tabacum) plants carrying an I-SceI endonuclease recognition site that, upon cleavage with I-SceI, generates DSB. Then, we retransformed these plants with two A. tumefaciens strains: one that allows transient expression of I-SceI to induce DSB and the other that carries a T-DNA with the I-SceI site and an integration selection marker. Integration of this latter T-DNA as full-length and I-SceI-digested molecules into the DSB site was analyzed in the resulting plants. Of 620 transgenic plants, 16 plants integrated T-DNA into DSB at their I-SceI sites; because DSB induces DNA repair, these results suggest that the invading T-DNA molecules target to the DNA repair sites for integration. Furthermore, of these 16 plants, seven plants incorporated T-DNA digested with I-SceI, which cleaves only double-stranded DNA. Thus, T-strand molecules can be converted into double-stranded intermediates before their integration into the DSB sites within the host cell genome.

Agrobacterium T-DNA Integration: Molecules and Models

Trends in Genetics : TIG. Aug, 2004  |  Pubmed ID: 15262410

Genetic transformation mediated by Agrobacterium involves the transfer of a DNA molecule (T-DNA) from the bacterium to the eukaryotic host cell, and its integration into the host genome. Whereas extensive work has revealed the biological mechanisms governing the production, Agrobacterium-to-plant cell transport and nuclear import of the Agrobacterium T-DNA, the integration step remains largely unexplored, although several different T-DNA integration mechanisms have been suggested. Recent genetic and functional studies have revealed the importance of host proteins involved in DNA repair and maintenance for T-DNA integration. In this article, we review our understanding of the specific function of these proteins and propose a detailed model for integration.

Involvement of Targeted Proteolysis in Plant Genetic Transformation by Agrobacterium

Nature. Sep, 2004  |  Pubmed ID: 15343337

Genetic transformation of plant cells by Agrobacterium represents a unique case of trans-kingdom DNA transfer. During this process, Agrobacterium exports its transferred (T) DNA and several virulence (Vir) proteins into the host cell, within which T-DNA nuclear import is mediated by VirD2 (ref. 3) and VirE2 (ref. 4) and their host cell interactors AtKAP-alpha and VIP1 (ref. 6), whereas its integration is mediated mainly by host cell proteins. The factors involved in the uncoating of T-DNA from its cognate proteins, which occurs before integration into the host genome, are still unknown. Here, we report that VirF-one of the few known exported Vir proteins whose function in the host cell remains unknown-is involved in targeted proteolysis of VIP1 and VirE2. We show that VirF localizes to the plant cell nucleus and interacts with VIP1, a nuclear protein. VirF, which contains an F-box motif, significantly destabilizes both VIP1 and VirE2 in yeast cells. Destabilization of VIP1 in the presence of VirF was then confirmed in planta. These results suggest that VIP1 and its cognate VirE2 are specifically targeted by the VirF-containing Skp1-Cdc53-cullin-F-box complex for proteolysis. The critical role of proteasomal degradation in Agrobacterium-mediated genetic transformation was also evident from inhibition of T-DNA expression by a proteasomal inhibitor.

Three-dimensional Reconstruction of Agrobacterium VirE2 Protein with Single-stranded DNA

The Journal of Biological Chemistry. Jun, 2004  |  Pubmed ID: 15054095

Agrobacterium tumefaciens infects plant cells by a unique mechanism involving an interkingdom genetic transfer. A single-stranded DNA substrate is transported across the two cell walls along with the bacterial virulence proteins VirD2 and VirE2. A single VirD2 molecule covalently binds to the 5'-end of the single-stranded DNA, while the VirE2 protein binds stoichiometrically along the length of the DNA, without sequence specificity. An earlier transmission/scanning transmission electron microscopy study indicated a solenoidal ("telephone coil") organization of the VirE2-DNA complex. Here we report a three-dimensional reconstruction of this complex using electron microscopy and single-particle image-processing methods. We find a hollow helical structure of 15.7-nm outer diameter, with a helical rise of 51.5 nm and 4.25 VirE2 proteins/turn. The inner face of the protein units contains a continuous wall and an inward protruding shelf. These structures appear to accommodate the DNA binding. Such a quaternary arrangement naturally sequesters the DNA from cytoplasmic nucleases and suggests a mechanism for its nuclear import by decoration with host cell factors. Coexisting with the helices, we also found VirE2 tetrameric ring structures. A two-dimensional average of the latter confirms the major features of the three-dimensional reconstruction.

Reorganization of Specific Chromosomal Domains and Activation of Silent Genes in Plant Cells Acquiring Pluripotentiality

Developmental Dynamics : an Official Publication of the American Association of Anatomists. May, 2004  |  Pubmed ID: 15108305

The transition from leaf cells to protoplasts (plant cells devoid of cell walls) confers pluripotentiality coupled with chromatin reorganization. Here, we sought to identify remodeled chromosomal domains in Arabidopsis protoplasts by tracking DNA sequences undergoing changes in DNA methylation and by identifying up-regulated genes. We observed a reduction in DNA methylation at a pericentromeric region of chromosome 1, and up-regulation of several members of the NAC (NAM/ATAF1/CUC2) domain family, two of which are located near the telomeric region of chromosome 1. Fluorescence in situ hybridization (FISH) analysis demonstrated that both pericentromeric and telomeric subdomains underwent chromatin decondensation. This decondensation is subdomain-specific inasmuch as centromeric repeats remained largely unchanged, whereas the 18S rDNA underwent condensation. Within the pericentromeric subdomain, VIP1, a gene encoding a b-Zip nuclear protein required for Agrobacterium infectivity, was transcriptionally activated. Overexpression of this gene in tobacco resulted in growth retardation and inhibition of differentiation and shoot formation. Altogether, our data indicate that acquisition of pluripotentiality involves changes in DNA methylation pattern and reorganization of specific chromosomal subdomains. This change leads to activation of silent genes whose products are involved in acquisition or maintenance of pluripotentiality and/or the ensuing fate of the cell.

Protein Interactions Involved in Nuclear Import of the Agrobacterium VirE2 Protein in Vivo and in Vitro

The Journal of Biological Chemistry. Jul, 2004  |  Pubmed ID: 15123622

Agrobacterium, the only known organism capable of trans-kingdom DNA transfer, genetically transforms plants by transferring a segment of its DNA, T-DNA, into the nucleus of the host cell where it integrates into the plant genome. One of the central events in this genetic transformation process is nuclear import of the T-DNA molecule, which to a large degree is mediated by the bacterial virulence protein VirE2. VirE2 is distinguished by its nuclear targeting, which occurs only in plant but not in animal cells and is facilitated by the cellular VIP1 protein. The molecular mechanism of the VIP1 function is still unclear. Here, we used in vitro assays for nuclear import and quantification of protein-protein interactions to directly demonstrate formation of ternary complexes between VirE2, VIP1, and a component of the cellular nuclear import machinery, karyopherin alpha. Our results indicate that VIP1 functions as a molecular bridge between VirE2 and karyopherin alpha, allowing VirE2 to utilize the host cell nuclear import machinery even without being directly recognized by its components.

High-throughput Fluorescent Tagging of Full-length Arabidopsis Gene Products in Planta

Plant Physiology. May, 2004  |  Pubmed ID: 15141064

We developed a high-throughput methodology, termed fluorescent tagging of full-length proteins (FTFLP), to analyze expression patterns and subcellular localization of Arabidopsis gene products in planta. Determination of these parameters is a logical first step in functional characterization of the approximately one-third of all known Arabidopsis genes that encode novel proteins of unknown function. Our FTFLP-based approach offers two significant advantages: first, it produces internally-tagged full-length proteins that are likely to exhibit native intracellular localization, and second, it yields information about the tissue specificity of gene expression by the use of native promoters. To demonstrate how FTFLP may be used for characterization of the Arabidopsis proteome, we tagged a series of known proteins with diverse subcellular targeting patterns as well as several proteins with unknown function and unassigned subcellular localization.

Functional Analysis of the Cucumber Mosaic Virus 2b Protein: Pathogenicity and Nuclear Localization

The Journal of General Virology. Oct, 2004  |  Pubmed ID: 15448377

The 2b protein encoded by Cucumber mosaic virus (CMV) has been shown to be a silencing suppressor and pathogenicity determinant in solanaceous hosts, but a movement determinant in cucumber. In addition, synergistic interactions between CMV and Zucchini yellow mosaic virus (ZYMV) have been described in several cucurbit species. Here, it was shown that deletion of the 2b gene from CMV prevented extensive systemic movement of the virus in zucchini squash, which could not be complemented by co-infection with ZYMV. Thus, ZYMV expressing a silencing suppressor with a different target could not complement the CMV 2b-specific movement function. Expression of the 2b protein from an attenuated ZYMV vector resulted in a synergistic response, largely restoring infection symptoms of wild-type ZYMV in several cucurbit species. Deletion or alteration of either of two nuclear localization signals (NLSs) did not affect nuclear localization in two assays, but did affect pathogenicity in several cucurbit species, whilst deletion of both NLSs led to loss of nuclear localization. The 2b protein interacted with an Arabidopsis thaliana karyopherin alpha protein (AtKAPalpha) in the yeast two-hybrid system, as did each of the two single NLS-deletion mutants. However, 2b protein containing a deletion of both NLSs was unable to interact with AtKAPalpha. These data suggest that the 2b protein localizes to the nucleus by using the karyopherin alpha-mediated system, but demonstrate that nuclear localization was insufficient for enhancement of the 2b-mediated pathogenic response in cucurbit hosts. Thus, the sequences corresponding to the two NLSs must have another role leading to pathogenicity enhancement.

The VirE3 Protein of Agrobacterium Mimics a Host Cell Function Required for Plant Genetic Transformation

The EMBO Journal. Jan, 2005  |  Pubmed ID: 15616576

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.

Control Improves with Age: Intercellular Transport in Plant Embryos and Adults

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2005  |  Pubmed ID: 15684080

PSAT Vectors: a Modular Series of Plasmids for Autofluorescent Protein Tagging and Expression of Multiple Genes in Plants

Plant Molecular Biology. Mar, 2005  |  Pubmed ID: 15821977

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.

Identification of an Interactor of Cadmium Ion-induced Glycine-rich Protein Involved in Regulation of Callose Levels in Plant Vasculature

Proceedings of the National Academy of Sciences of the United States of America. Aug, 2005  |  Pubmed ID: 16103368

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.

Uncoupling of the Functions of the Arabidopsis VIP1 Protein in Transient and Stable Plant Genetic Transformation by Agrobacterium

Proceedings of the National Academy of Sciences of the United States of America. Apr, 2005  |  Pubmed ID: 15824315

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.

Effects of Calreticulin on Viral Cell-to-cell Movement

Plant Physiology. Aug, 2005  |  Pubmed ID: 16006596

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.

The Plant VirE2 Interacting Protein 1. a Molecular Link Between the Agrobacterium T-complex and the Host Cell Chromatin?

Plant Physiology. Jul, 2005  |  Pubmed ID: 16010006

Plant Viruses. Invaders of Cells and Pirates of Cellular Pathways

Plant Physiology. Aug, 2005  |  Pubmed ID: 16172093

Involvement of KU80 in T-DNA Integration in Plant Cells

Proceedings of the National Academy of Sciences of the United States of America. Dec, 2005  |  Pubmed ID: 16380432

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.

Nuclear Export of African Swine Fever Virus P37 Protein Occurs Through Two Distinct Pathways and is Mediated by Three Independent Signals

Journal of Virology. Feb, 2006  |  Pubmed ID: 16415017

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.

Agrobacterium-mediated Genetic Transformation of Plants: Biology and Biotechnology

Current Opinion in Biotechnology. Apr, 2006  |  Pubmed ID: 16459071

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.

Pollen-specific Pectin Methylesterase Involved in Pollen Tube Growth

Developmental Biology. Jun, 2006  |  Pubmed ID: 16564517

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.

Will You Let Me Use Your Nucleus? How Agrobacterium Gets Its T-DNA Expressed in the Host Plant Cell

Canadian Journal of Physiology and Pharmacology. Mar-Apr, 2006  |  Pubmed ID: 16902581

Agrobacterium is the only known bacterium capable of natural DNA transfer into a eukaryotic host. The genes transferred to host plants are contained on a T-DNA (transferred DNA) molecule, the transfer of which begins with its translocation, along with several effector proteins, from the bacterial cell to the host-cell cytoplasm. In the host cytoplasm, the T-complex is formed from a single-stranded copy of the T-DNA (T-strand) associated with several bacterial and host proteins and it is imported into the host nucleus via interactions with the host nuclear import machinery. Once inside the nucleus, the T-complex is most likely directed to the host genome by associating with histones. Finally, the chromatin-associated T-complex is uncoated from its escorting proteins prior to the conversion of the T-strand to a double-stranded form and its integration into the host genome.

Subcellular Localization of Interacting Proteins by Bimolecular Fluorescence Complementation in Planta

Journal of Molecular Biology. Oct, 2006  |  Pubmed ID: 16949607

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.

A Case of Promiscuity: Agrobacterium's Endless Hunt for New Partners

Trends in Genetics : TIG. Jan, 2006  |  Pubmed ID: 16289425

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.

Nuclear Import and Export of Plant Virus Proteins and Genomes

Molecular Plant Pathology. Mar, 2006  |  Pubmed ID: 20507434

SUMMARY Nuclear import and export are crucial processes for any eukaryotic cell, as they govern substrate exchange between the nucleus and the cytoplasm. Proteins involved in the nuclear transport network are generally conserved among eukaryotes, from yeast and fungi to animals and plants. Various pathogens, including some plant viruses, need to enter the host nucleus to gain access to its replication machinery or to integrate their DNA into the host genome; the newly replicated viral genomes then need to exit the nucleus to spread between host cells. To gain the ability to enter and exit the nucleus, these pathogens encode proteins that recognize cellular nuclear transport receptors and utilize the host's nuclear import and export pathways. Here, we review and discuss our current knowledge about the molecular mechanisms by which plant viruses find their way into and out of the host cell nucleus.

Mammalian Cells

Methods in Molecular Biology (Clifton, N.J.). 2006  |  Pubmed ID: 17033084

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.

How Pollen Tubes Grow

Developmental Biology. Mar, 2007  |  Pubmed ID: 17214979

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.

Biological Systems of the Host Cell Involved in Agrobacterium Infection

Cellular Microbiology. Jan, 2007  |  Pubmed ID: 17222189

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.

C2H2 Zinc Finger-SET Histone Methyltransferase is a Plant-specific Chromatin Modifier

Developmental Biology. Mar, 2007  |  Pubmed ID: 17224141

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.

Arabidopsis VIRE2 INTERACTING PROTEIN2 is Required for Agrobacterium T-DNA Integration in Plants

The Plant Cell. May, 2007  |  Pubmed ID: 17496122

Agrobacterium tumefaciens-mediated genetic transformation is an efficient tool for genetic engineering of plants. VirE2 is a single-stranded DNA binding Agrobacterium protein that is transported into the plant cell and presumably protects the T-DNA from degradation. Using a yeast two-hybrid system, we identified Arabidopsis thaliana VIRE2-INTERACTING PROTEIN2 (VIP2) with a NOT domain that is conserved in both plants and animals. Furthermore, we provide evidence supporting VIP2 interaction with VIP1, a basic domain/leucine zipper motif-containing protein required for nuclear import and integration of T-DNA. Virus-induced gene silencing of VIP2 in Nicotiana benthamiana and characterization of the Arabidopsis vip2 mutant (At vip2) demonstrate that VIP2 is required for Agrobacterium-mediated stable transformation but not for transient transformation. Assays based upon a promoter-trap vector and quantification of T-DNA integration further confirmed VIP2 involvement in T-DNA integration. Interestingly, VIP2 transcripts were induced to a greater extent over prolonged periods after infection with a T-DNA transfer-competent Agrobacterium strain compared with the transfer-deficient Agrobacterium strain. Transcriptome analyses of At vip2 suggest that VIP2 is likely a transcriptional regulator, and the recalcitrancy to transformation in At vip2 is probably due to the combination of muted gene expression response upon Agrobacterium infection and repression of histone genes resulting in decreased T-DNA integration events.

PSITE Vectors for Stable Integration or Transient Expression of Autofluorescent Protein Fusions in Plants: Probing Nicotiana Benthamiana-virus Interactions

Molecular Plant-microbe Interactions : MPMI. Jul, 2007  |  Pubmed ID: 17601162

Plant functional proteomics research is increasingly dependent upon vectors that facilitate high-throughput gene cloning and expression of fusions to autofluorescent proteins. Here, we describe the pSITE family of plasmids, a new set of Agrobacterium binary vectors, suitable for the stable integration or transient expression of various autofluorescent protein fusions in plant cells. The pSITE vectors permit single-step Gateway-mediated recombination cloning for construction of binary vectors that can be used directly in transient expression studies or for the selection of transgenic plants on media containing kanamycin. These vectors can be used to express native proteins or fusions to monmeric red fluorescent protein or the enhanced green fluorescent protein and its cyan and yellow-shifted spectral variants. We have validated the vectors for use in transient expression assays and for the generation of transgenic plants. Additionally, we have generated markers for fluorescent highlighting of actin filaments, chromatin, endoplasmic reticulum, and nucleoli. Finally, we show that pSITE vectors can be used for targeted gene expression in virus-infected cells, which should facilitate high-throughput characterization of protein dynamics in host-virus interactions.

Yeast-plant Coupled Vector System for Identification of Nuclear Proteins

Plant Physiology. Dec, 2007  |  Pubmed ID: 17704231

Nuclear proteins are involved in many critical biological processes within plant cells and, therefore, are in the focus of studies that usually begin with demonstrating that the protein of interest indeed exhibits nuclear localization. Thus, studies of plant nuclear proteins would be facilitated by a convenient experimental system for identification of proteins that are actively imported into the cell nucleus and visualization of their nuclear accumulation in vivo. To this end, we developed a system of vectors that allows screening for cDNAs coding for nuclear proteins in a simple genetic assay in yeast cells, and verification of nuclear accumulation in planta following one-step transfer and autofluorescent tagging of the identified clones into a multiple cloning site-compatible and reading frame-compatible plant expression vector. In a recommended third experimental step, the plant expression cassette containing the identified clone can be transferred, also by a one-step cloning, into a binary multigene expression vector for transient or stable coexpression with any other proteins.

Advanced Expression Vector Systems: New Weapons for Plant Research and Biotechnology

Plant Physiology. Dec, 2007  |  Pubmed ID: 18056858

Arabidopsis Co-repressor Complexes Containing Polyamine Oxidase-like Proteins and Plant-specific Histone Methyltransferases

Plant Signaling & Behavior. May, 2007  |  Pubmed ID: 19704688

Regulation of genes by repression of transcription represents a virtually universal mechanism that underlies such diverse biological processes as restriction of expression of neuronal genes to neurons in mammals, and control of flowering in plants. However, while the molecular mechanisms of transcriptional gene silencing in animal systems are being intensively studied, our understanding of these processes in plants is very sparse and, because plants often utilize unique strategies to establish and maintain chromatin states, only limited use can be made of information available on epigenetic modifications in nonplant systems.

Agrobacterium-mediated Genetic Transformation of Tea Leaf Explants: Effects of Counteracting Bactericidity of Leaf Polyphenols Without Loss of Bacterial Virulence

Plant Cell Reports. Feb, 2007  |  Pubmed ID: 16972098

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.

Suppressor of RNA Silencing Encoded by Tomato Yellow Leaf Curl Virus-Israel

Virology. Feb, 2007  |  Pubmed ID: 16979684

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.

Interaction with Host SGS3 is Required for Suppression of RNA Silencing by Tomato Yellow Leaf Curl Virus V2 Protein

Proceedings of the National Academy of Sciences of the United States of America. Jan, 2008  |  Pubmed ID: 18165314

The V2 protein of tomato yellow leaf curl geminivirus (TYLCV) functions as an RNA-silencing suppressor that counteracts the innate immune response of the host plant. The host-cell target of V2, however, remains unknown. Here we show that V2 interacts directly with SlSGS3, the tomato homolog of the Arabidopsis SGS3 protein (AtSGS3), which is known to be involved in the RNA-silencing pathway. SlSGS3 genetically complemented an AtSGS3 mutation and restored RNA silencing, indicating that SlSGS3 is indeed a functional homolog of AtSGS3. A point mutant of V2 that is unable to bind SlSGS3 also lost its ability to suppress RNA silencing, suggesting a correlation between the V2-SlSGS3 interaction in planta and the suppressor activity of V2.

African Swine Fever Virus P10 Protein Exhibits Nuclear Import Capacity and Accumulates in the Nucleus During Viral Infection

Veterinary Microbiology. Jul, 2008  |  Pubmed ID: 18243588

African swine fever virus (ASFV), a large enveloped DNA-containing virus, infects domestic and wild pigs, and multiplies in soft ticks, causing an economically relevant hemorrhagic disease. Evaluation of the nuclear import ability of ASFV p10 protein was the major purpose of the present work. Two approaches were used to determine if p10 protein is imported into the nucleus by an active process: a yeast-based nuclear import assay and the determination of the subcellular localization of p10 protein in mammalian cells by fluorescence microscopy. The results obtained clearly demonstrate that p10 protein is actively imported into the nucleus, both in yeast and mammalian cells. Experiments aiming at identifying the critical residues responsible for the nuclear import of ASFV p10 protein indicate that the amino acids comprised between the positions 71 and 77 are important, although not sufficient, for the protein active nuclear import. In ASFV-infected cells, the p10 protein strongly accumulates in the nucleus at late times post-infection, indicating that p10 protein may accomplish an important function inside the nucleus during the late phase of the viral replication cycle.

Probing Interactions Between Plant Virus Movement Proteins and Nucleic Acids

Methods in Molecular Biology (Clifton, N.J.). 2008  |  Pubmed ID: 18370264

Most plant viruses move between plant cells with the help of their movement proteins (MPs). MPs are multifunctional proteins, and one of their functions is almost invariably binding to nucleic acids. Presumably, the MP-nucleic acid interaction is directly involved in formation of nucleoprotein complexes that function as intermediates in the cell-to-cell transport of many plant viruses. Thus, when studying a viral MP, it is important to determine whether or not it binds nucleic acids, and to characterize the hallmark parameters of such binding, i.e., preference for single- or double-stranded nucleic acids and binding cooperativity and sequence specificity. Here, we present two major experimental approaches, native gel mobility shift assay and ultra violet (UV) light cross-linking, for detection and characterization of MP binding to DNA and RNA molecules. We also describe protocols for purification of recombinant viral MPs over-expressed in bacteria and production of different DNA and RNA probes for these binding assays.

Agrobacterium Tumefaciens-induced Bacteraemia Does Not Lead to Reporter Gene Expression in Mouse Organs

PloS One. 2008  |  Pubmed ID: 18523638

Agrobacterium tumefaciens is the main plant biotechnology gene transfer tool with host range which can be extended to non-plant eukaryotic organisms under laboratory conditions. Known medical cases of Agrobacterium species isolation from bloodstream infections necessitate the assessment of biosafety-related risks of A. tumefaciens encounters with mammalian organisms. Here, we studied the survival of A. tumefaciens in bloodstream of mice injected with bacterial cultures. Bacterial titers of 10(8) CFU were detected in the blood of the injected animals up to two weeks after intravenous injection. Agrobacteria carrying Cauliflower mosaic virus (CaMV) 35S promoter-based constructs and isolated from the injected mice retained their capacity to promote green fluorescent protein (GFP) synthesis in Nicotiana benthamiana leaves. To examine whether or not the injected agrobacteria are able to express in mouse organs, we used an intron-containing GFP (GFPi) reporter driven either by a cytomegalovirus (CMV) promoter or by a CaMV 35S promoter. Western and northern blot analyses as well as RT-PCR analysis of liver, spleen and lung of mice injected with A. tumefaciens detected neither GFP protein nor its transcripts. Thus, bacteraemia induced in mice by A. tumefaciens does not lead to detectable levels of genetic transformation of mouse organs.

Localizing Protein-protein Interactions by Bimolecular Fluorescence Complementation in Planta

Methods (San Diego, Calif.). Jul, 2008  |  Pubmed ID: 18586107

The application of novel assays for basic cell research is tightly linked to the development of easy-to-use and versatile tools and protocols for implementing such technologies for a wide range of applications and model species. The bimolecular fluorescence complementation (BiFC) assay is one such novel method for which tools and protocols for its application in plant cell research are still being developed. BiFC is a powerful tool which enables not only detection, but also visualization and subcellular localization of protein-protein interactions in living cells. Here we describe the application of BiFC in plant cells while focusing on the use of our versatile set of vectors which were specifically designed to facilitate the transformation, expression and imaging of protein-protein interactions in various plant species. We discuss the considerations of using our system in various plant model systems, the use of single versus multiple expression cassettes, the application of our vectors using various transformation methods and the use of internal fluorescent markers which can assist in signal localization and easy data acquisition in living cells.

Association of the Agrobacterium T-DNA-protein Complex with Plant Nucleosomes

Proceedings of the National Academy of Sciences of the United States of America. Oct, 2008  |  Pubmed ID: 18832163

Agrobacterium represents the only natural example of transkingdom transfer of genetic information, from bacteria to plants. Before the bacterial transferred DNA (T-DNA) can integrate into the plant genome, it should be targeted to and bind the host chromatin. However, the T-DNA association with the host chromatin has not been demonstrated. Here, we study T-DNA binding to plant nucleosomes in vitro and show that it is mediated by bacterial and host proteins associated with the T-DNA. The main factor that determines nucleosomal binding of the T-DNA is the cellular VirE2-interacting protein 1 (VIP1), which functions as a molecular link between the T-DNA-associated bacterial virulence protein VirE2 and core histones. The presence of both VIP1 and VirE2 is required for association of the T-DNA with mononucleosomes in which the DNA molecule exists as a tripartite complex DNA-VirE2-VIP1. Furthermore, this nucleosome-associated ternary complex can bind another bacterial virulence factor, VirF, which is an F-box protein known to target both VirE2 and VIP1 for proteasomal degradation and uncoat the T-DNA.

Recent Patents on Agrobacterium-mediated Gene and Protein Transfer, for Research and Biotechnology

Recent Patents on DNA & Gene Sequences. 2008  |  Pubmed ID: 19075947

Agrobacterium has been widely used, in the last decades, for genetic transformation of a large number of plant species, and the genes and DNA sequences involved in this process have been subject of numerous patents. This review focuses on recent discoveries, which have shown new possibilities for the utilization of this versatile microorganism. For example, the identification of an ever-increasing number of the bacterial and plant factors involved in the Agrobacterium-mediated DNA transfer and integration may lead to new applications in various fields of research and biotechnology. One of the main challenges in the Agrobacterium-mediated gene transfer technology is to achieve a better control of the integration and expression of transferred genes in the host cells and to apply it for targeted integration into the host genome or gene replacement (a technique not yet available in plants). In addition to genetic transformation of plants, under laboratory conditions, the host range of Agrobacterium can be extended to virtually all eukaryotic species, as demonstrated for various fungi, sea urchins, and animal cells. Not only can Agrobacterium transfer DNA to these very diverse hosts, but also its virulence machinery is able to inject proteins into the host cell, independently of the DNA transfer. Thus, Agrobacterium represents a universal gene and protein transfer machine.

Regulation of Root Elongation by Histone Acetylation in Arabidopsis

Journal of Molecular Biology. Jan, 2009  |  Pubmed ID: 18835563

Transcriptional repression by histone modification represents a universal mechanism that underlies critical biological processes, such as neurogenesis and hematopoietic differentiation, in animals. In plants, however, the extent to which these regulatory pathways are involved in development and morphogenesis is not well defined. SWP1/LDL1 is a component of a plant corepressor complex that is involved in regulation of flower timing. Here, we report that SWP1 also plays a role in the regulation of root elongation by repressing a root-specific gene lateral root primordium 1 (LRP1) via histone deacetylation. A null mutation in SWP1 results in hyperacetylation of histones H3 and H4 in LRP1 chromatin, elevation of LRP1 expression, and increased root elongation. This effect of SWP1 knockout on the root phenotype is mimicked by transgenic expression of LRP1, which bypasses the SWP1-mediated suppression of the native gene. Thus, SWP1 likely functions as a regulator of developmental events both in the shoot and in the root meristem.

The Small Subunit of Snapdragon Geranyl Diphosphate Synthase Modifies the Chain Length Specificity of Tobacco Geranylgeranyl Diphosphate Synthase in Planta

The Plant Cell. Dec, 2009  |  Pubmed ID: 20028839

Geranyl diphosphate (GPP), the precursor of many monoterpene end products, is synthesized in plastids by a condensation of dimethylallyl diphosphate and isopentenyl diphosphate (IPP) in a reaction catalyzed by homodimeric or heterodimeric GPP synthase (GPPS). In the heterodimeric enzymes, a noncatalytic small subunit (GPPS.SSU) determines the product specificity of the catalytic large subunit, which may be either an active geranylgeranyl diphosphate synthase (GGPPS) or an inactive GGPPS-like protein. Here, we show that expression of snapdragon (Antirrhinum majus) GPPS.SSU in tobacco (Nicotiana tabacum) plants increased the total GPPS activity and monoterpene emission from leaves and flowers, indicating that the introduced catalytically inactive GPPS.SSU found endogenous large subunit partner(s) and formed an active snapdragon/tobacco GPPS in planta. Bimolecular fluorescence complementation and in vitro enzyme analysis of individual and hybrid proteins revealed that two of four GGPPS-like candidates from tobacco EST databases encode bona fide GGPPS that can interact with snapdragon GPPS.SSU and form a functional GPPS enzyme in plastids. The formation of chimeric GPPS in transgenic plants also resulted in leaf chlorosis, increased light sensitivity, and dwarfism due to decreased levels of chlorophylls, carotenoids, and gibberellins. In addition, these transgenic plants had reduced levels of sesquiterpene emission, suggesting that the export of isoprenoid intermediates from the plastids into the cytosol was decreased. These results provide genetic evidence that GPPS.SSU modifies the chain length specificity of phylogenetically distant GGPPS and can modulate IPP flux distribution between GPP and GGPP synthesis in planta.

Functional Transient Genetic Transformation of Arabidopsis Leaves by Biolistic Bombardment

Nature Protocols. 2009  |  Pubmed ID: 19131958

Transient gene expression is an indispensable tool for studying functions of gene products. In the case of plants, transient introduction of genes by Agrobacterium infiltration is a method of choice for many species. However, this technique does not work efficiently in Arabidopsis leaf tissue, the most widely used model system for basic plant biology research. Here we present an optimized protocol for biolistic delivery of plasmid DNA into the epidermis of Arabidopsis leaves, which can be easily performed using the Bio-Rad Helios gene gun system. This protocol yields efficient and reproducible transient expression of diverse genes and is exemplified here for use in a functional assay of a transcription repressor and for the subcellular localization and cell-to-cell movement of plant viral movement protein. This protocol is suitable for studies of biological function and subcellular localization of the gene product of interest directly in planta by utilizing different types of activity-based assays. Using this procedure, the data are obtained after 2-4 d of work.

Proteasomal Degradation in Plant-pathogen Interactions

Seminars in Cell & Developmental Biology. Dec, 2009  |  Pubmed ID: 19505586

The ubiquitin/26S proteasome pathway is a basic biological mechanism involved in the regulation of a multitude of cellular processes. Increasing evidence indicates that plants utilize the ubiquitin/26S proteasome pathway in their immune response to pathogen invasion, emphasizing the role of this pathway during plant-pathogen interactions. The specific functions of proteasomal degradation in plant-pathogen interactions are diverse, and do not always benefit the host plant. Although in some cases, proteasomal degradation serves as an effective barrier to help plants ward off pathogens, in others, it is used by the pathogen to enhance the infection process. This review discusses the different roles of the ubiquitin/26S proteasome pathway during interactions of plants with pathogenic viruses, bacteria, and fungi.

Agrobacterium Aiming for the Host Chromatin: Host and Bacterial Proteins Involved in Interactions Between T-DNA and Plant Nucleosomes

Communicative & Integrative Biology. 2009  |  Pubmed ID: 19513263

Agrobacterium genetically transforms its hosts by transferring a segment of DNA (T-DNA) into the host cell and integrating it into the host genome. Integration requires a close interaction between T-DNA, which is packaged into a nucleoprotein complex (T-complex) by bacterial virulence (Vir) proteins, and the host chromatin. This interaction is facilitated by the host protein VIP 1, which binds both to the major protein component of the T-complex, VirE2, and to the core histones. Recently, VIP1 has been demonstrated to mediate the interaction between plant nucleosomes and VirE2-DNA complexes (i.e., synthetic T-complex-like structures) in vitro. Here, we discuss major implications of these observations-such as the possible role of core histone modifications, proteasomal uncoating of the T-complex mediated by the bacterial F-box protein VirF, and the need for changes in chromatin structure to render it accessible to the T-DNA integration-for the process of chromatin targeting of foreign DNA and its integration into the eukaryotic genome.

Systems Biology of Plant-pathogen Interactions

Seminars in Cell & Developmental Biology. Dec, 2009  |  Pubmed ID: 19501663

The Roles of Plant Phenolics in Defence and Communication During Agrobacterium and Rhizobium Infection

Molecular Plant Pathology. Sep, 2010  |  Pubmed ID: 20696007

Phenolics are aromatic benzene ring compounds with one or more hydroxyl groups produced by plants mainly for protection against stress. The functions of phenolic compounds in plant physiology and interactions with biotic and abiotic environments are difficult to overestimate. Phenolics play important roles in plant development, particularly in lignin and pigment biosynthesis. They also provide structural integrity and scaffolding support to plants. Importantly, phenolic phytoalexins, secreted by wounded or otherwise perturbed plants, repel or kill many microorganisms, and some pathogens can counteract or nullify these defences or even subvert them to their own advantage. In this review, we discuss the roles of phenolics in the interactions of plants with Agrobacterium and Rhizobium.

Agrobacterium Induces Expression of a Host F-box Protein Required for Tumorigenicity

Cell Host & Microbe. Mar, 2010  |  Pubmed ID: 20227663

Agrobacterium exports DNA into plant cells, eliciting neoplastic growths on many plant species. During this process, a Skp1-Cdc53-cullin-F-box (SCF) complex that contains the bacterial virulence F-box protein VirF facilitates genetic transformation by targeting for proteolysis proteins, the Agrobacterium protein VirE2 and the host protein VIP1, that coat the transferred DNA. However, some plant species do not require VirF for transformation. Here, we show that Agrobacterium induces expression of a plant F-box protein, which we designated VBF for VIP1-binding F-box protein, that can functionally replace VirF, regulating levels of the VirE2 and VIP1 proteins via a VBF-containing SCF complex. When expressed in Agrobacterium and exported into the plant cell, VBF functionally complements tumor formation by a strain lacking VirF. VBF expression is known to be induced by diverse pathogens, suggesting that Agrobacterium has co-opted a plant defense response and that bacterial VirF and plant VBF both contribute to targeted proteolysis that promotes plant genetic transformation.

Plant Defense Pathways Subverted by Agrobacterium for Genetic Transformation

Plant Signaling & Behavior. Oct, 2010  |  Pubmed ID: 20890133

The soil phytopathogen Agrobacterium has the unique ability to introduce single-stranded transferred DNA (T-DNA) from its tumor-inducing (Ti) plasmid into the host cell in a process known as horizontal gene transfer. Following its entry into the host cell cytoplasm, the T-DNA associates with the bacterial virulence (Vir) E2 protein, also exported from Agrobacterium, creating the T-DNA nucleoprotein complex (T-complex), which is then translocated into the nucleus where the DNA is integrated into the host chromatin. VirE2 protects the T-DNA from the host DNase activities, packages it into a helical filament, and interacts with the host proteins, one of which, VIP1, facilitates nuclear import of the T-complex and its subsequent targeting to the host chromatin. Although the VirE2 and VIP1 protein components of the T-complex are vital for its intracellular transport, they must be removed to expose the T-DNA for integration. Our recent work demonstrated that this task is aided by an host defense-related F-box protein VBF that is induced by Agrobacterium infection and that recognizes and binds VIP1. VBF destabilizes VirE2 and VIP1 in yeast and plant cells, presumably via SCF-mediated proteasomal degradation. VBF expression in and export from the Agrobacterium cell lead to increased tumorigenesis. Here, we discuss these findings in the context of the "arms race" between Agrobacterium infectivity and plant defense.

Pol II-directed Short RNAs Suppress the Nuclear Export of MRNA

Plant Molecular Biology. Dec, 2010  |  Pubmed ID: 20953971

The synthesis and subsequent nuclear export of non-coding RNA (ncRNA) directed by RNA polymerase (Pol) II is very sensitive to abiotic and biotic external stimuli including pathogen challenges. To assess whether stress-induced ncRNAs may suppress the nuclear export of mRNA, we exploited the ability of Agrobacterium tumefaciens to co-deliver Pol I, II and III promoter-based vectors for the transcription of short (s) ncRNAs, GFP mRNA or genomic RNA of plant viruses (Tobacco mosaic virus, TMV; or Potato virus X, PVX) into the nucleus of Nicotiana benthamiana cells. We showed that, in contrast to Pol I- and Pol III-derived sncRNAs, all tested Pol II-derived sncRNAs (U6 RNA, tRNA or artificial RNAs) resulted in decreased expression of GFP and host mRNA. The level of this inhibitory effect depended on the non-coding transcript length and promoter strength. Short coding RNA (scRNA) can also compete with mRNA for nuclear export. We showed that scRNA, an artificial 117-nt short sequence encoding Elastin-Like peptide element tandems with FLAG sequence (ELF) and the 318-nt N. benthamiana antimicrobial peptide thionin (defensin) gene efficiently decreased GFP expression. The stress-induced export of Pol II-derived sncRNA and scRNA into the cytoplasm via the mRNA export pathway may block nucleocytoplasmic traffic including the export of mRNA responsible for antivirus protection. Consistent with this model, we observed that Pol II-derived sncRNAs as well as scRNA, thionin and ELF strongly enhanced the cytoplasmic reproduction of TMV and PVX RNA.

Autoluminescent Plants

PloS One. 2010  |  Pubmed ID: 21103397

Prospects of obtaining plants glowing in the dark have captivated the imagination of scientists and layman alike. While light emission has been developed into a useful marker of gene expression, bioluminescence in plants remained dependent on externally supplied substrate. Evolutionary conservation of the prokaryotic gene expression machinery enabled expression of the six genes of the lux operon in chloroplasts yielding plants that are capable of autonomous light emission. This work demonstrates that complex metabolic pathways of prokaryotes can be reconstructed and function in plant chloroplasts and that transplastomic plants can emit light that is visible by naked eye.

ANK, a Host Cytoplasmic Receptor for the Tobacco Mosaic Virus Cell-to-cell Movement Protein, Facilitates Intercellular Transport Through Plasmodesmata

PLoS Pathogens. 2010  |  Pubmed ID: 21124937

Plasmodesma (PD) is a channel structure that spans the cell wall and provides symplastic connection between adjacent cells. Various macromolecules are known to be transported through PD in a highly regulated manner, and plant viruses utilize their movement proteins (MPs) to gate the PD to spread cell-to-cell. The mechanism by which MP modifies PD to enable intercelluar traffic remains obscure, due to the lack of knowledge about the host factors that mediate the process. Here, we describe the functional interaction between Tobacco mosaic virus (TMV) MP and a plant factor, an ankyrin repeat containing protein (ANK), during the viral cell-to-cell movement. We utilized a reverse genetics approach to gain insight into the possible involvement of ANK in viral movement. To this end, ANK overexpressor and suppressor lines were generated, and the movement of MP was tested. MP movement was facilitated in the ANK-overexpressing plants, and reduced in the ANK-suppressing plants, demonstrating that ANK is a host factor that facilitates MP cell-to-cell movement. Also, the TMV local infection was largely delayed in the ANK-suppressing lines, while enhanced in the ANK-overexpressing lines, showing that ANK is crucially involved in the infection process. Importantly, MP interacted with ANK at PD. Finally, simultaneous expression of MP and ANK markedly decreased the PD levels of callose, β-1,3-glucan, which is known to act as a molecular sphincter for PD. Thus, the MP-ANK interaction results in the downregulation of callose and increased cell-to-cell movement of the viral protein. These findings suggest that ANK represents a host cellular receptor exploited by MP to aid viral movement by gating PD through relaxation of their callose sphincters.

Epigenetic Control of Agrobacterium T-DNA Integration

Biochimica Et Biophysica Acta. Aug, 2011  |  Pubmed ID: 21296691

To genetically transform plants, Agrobacterium transfers its T-DNA into the host cell and integrates it into the plant genome, resulting in neoplastic growths. Over the past 2 decades, a great deal has been learned about the molecular mechanism by which Agrobacterium produces T-DNA and transports it into the host nucleus. However, T-DNA integration, which is the limiting, hence, the most critical step of the transformation process, largely remains an enigma. Increasing evidence suggests that Agrobacterium utilizes the host DNA repair machinery to facilitate T-DNA integration. Meanwhile, it is well known that chromatin modifications, including the phosphorylation of histone H2AX, play an important role in DNA repair. Thus, by implication, such epigenetic codes in chromatin may also have a considerable impact on T-DNA integration, although the direct evidence to demonstrate this hypothesis is still lacking. In this review, we summarize the recent advances in our understanding of Agrobacterium T-DNA integration and discuss the potential link between this process and the epigenetic information in the host chromatin. This article is part of a Special Issue entitled: Epigenetic Control of cellular and developmental processes in plants.

Involvement of KDM1C Histone Demethylase-OTLD1 Otubain-like Histone Deubiquitinase Complexes in Plant Gene Repression

Proceedings of the National Academy of Sciences of the United States of America. Jul, 2011  |  Pubmed ID: 21690391

Covalent modifications of histones, such as acetylation, methylation and ubiquitination, are central for regulation of gene expression. Heterochromatic gene silencing, for example, is associated with hypoacetylation, methylation and demethylation, and deubiquitination of specific amino acid residues in histone molecules. Many of these changes can be effected by histone-modifying repressor complexes that include histone lysine demethylases, such as KDM1 in animals and KDM1C in plants. However, whereas KDM1-containing repressor complexes have been implicated in histone demethylation, methylation and deacetylation, whether or not they can also mediate histone deubiquitination remains unknown. We identify an Arabidopsis otubain-like deubiquitinase OTLD1 which directly interacts with the Arabidopsis KDM1C in planta, and use one target gene to exemplify that both OTLD1 and KDM1C are involved in transcriptional gene repression via histone deubiquitination and demethylation. We also show that OTLD1 binds plant chromatin and has enzymatic histone deubiquitinase activity, specific for the H2B histone. Thus, we suggest that, during gene repression, lysine demethylases can directly interact and function in a protein complex with histone deubiquitinases.

To Gate, or Not to Gate: Regulatory Mechanisms for Intercellular Protein Transport and Virus Movement in Plants

Molecular Plant. Sep, 2011  |  Pubmed ID: 21746703

Cell-to-cell signal transduction is vital for orchestrating the whole-body physiology of multi-cellular organisms, and many endogenous macromolecules, proteins, and nucleic acids function as such transported signals. In plants, many of these molecules are transported through plasmodesmata (Pd), the cell wall-spanning channel structures that interconnect plant cells. Furthermore, Pd also act as conduits for cell-to-cell movement of most plant viruses that have evolved to pirate these channels to spread the infection. Pd transport is presumed to be highly selective, and only a limited repertoire of molecules is transported through these channels. Recent studies have begun to unravel mechanisms that actively regulate the opening of the Pd channel to allow traffic. This macromolecular transport between cells comprises two consecutive steps: intracellular targeting to Pd and translocation through the channel to the adjacent cell. Here, we review the current knowledge of molecular species that are transported though Pd and the mechanisms that control this traffic. Generally, Pd traffic can occur by passive diffusion through the trans-Pd cytoplasm or through the membrane/lumen of the trans-Pd ER, or by active transport that includes protein-protein interactions. It is this latter mode of Pd transport that is involved in intercellular traffic of most signal molecules and is regulated by distinct and sometimes interdependent mechanisms, which represent the focus of this article.

Extracellular VirB5 Enhances T-DNA Transfer from Agrobacterium to the Host Plant

PloS One. 2011  |  Pubmed ID: 22028781

VirB5 is a type 4 secretion system protein of Agrobacterium located on the surface of the bacterial cell. This localization pattern suggests a function for VirB5 which is beyond its known role in biogenesis and/or stabilization of the T-pilus and which may involve early interactions between Agrobacterium and the host cell. Here, we identify VirB5 as the first Agrobacterium virulence protein that can enhance infectivity extracellularly. Specifically, we show that elevating the amounts of the extracellular VirB5--by exogenous addition of the purified protein, its overexpression in the bacterium, or transgenic expression in and secretion out of the host cell--enhances the efficiency the Agrobacterium-mediated T-DNA transfer, as measured by transient expression of genes contained on the transferred T-DNA molecule. Importantly, the exogenous VirB5 enhanced transient T-DNA expression in sugar beet, a major crop recalcitrant to genetic manipulation. Increasing the pool of the extracellular VirB5 did not complement an Agrobacterium virB5 mutant, suggesting a dual function for VirB5: in the bacterium and at the bacterium-host cell interface. Consistent with this idea, VirB5 expressed in the host cell, but not secreted, had no effect on the transformation efficiency. That the increase in T-DNA expression promoted by the exogenous VirB5 was not due to its effects on bacterial growth, virulence gene induction, bacterial attachment to plant tissue, or host cell defense response suggests that VirB5 participates in the early steps of the T-DNA transfer to the plant cell.

Biology of Callose (β-1,3-glucan) Turnover at Plasmodesmata

Protoplasma. Jan, 2011  |  Pubmed ID: 21116665

The turnover of callose (β-1,3-glucan) within cell walls is an essential process affecting many developmental, physiological and stress related processes in plants. The deposition and degradation of callose at the neck region of plasmodesmata (Pd) is one of the cellular control mechanisms regulating Pd permeability during both abiotic and biotic stresses. Callose accumulation at Pd is controlled by callose synthases (CalS; EC 2.4.1.34), endogenous enzymes mediating callose synthesis, and by β-1,3-glucanases (BG; EC 3.2.1.39), hydrolytic enzymes which specifically degrade callose. Transcriptional and posttranslational regulation of some CalSs and BGs are strongly controlled by stress signaling, such as that resulting from pathogen invasion. We review the role of Pd-associated callose in the regulation of intercellular communication during developmental, physiological, and stress response processes. Special emphasis is placed on the involvement of Pd-callose in viral pathogenicity. Callose accumulation at Pd restricts virus movement in both compatible and incompatible interactions, while its degradation promotes pathogen spread. Hence, studies on mechanisms of callose turnover at Pd during viral cell-to-cell spread are of importance for our understanding of host mechanisms exploited by viruses in order to successfully spread within the infected plant.

Agrobacterium Counteracts Host-induced Degradation of Its Effector F-box Protein

Science Signaling. Oct, 2011  |  Pubmed ID: 22009152

The SCF (Skp1-Cul1-F-box protein) ubiquitin ligase complex plays a pivotal role in various biological processes, including host-pathogen interactions. Many pathogens exploit the host SCF machinery to promote efficient infection by translocating pathogen-encoded F-box proteins into the host cell. How pathogens ensure sufficient amounts of the F-box effectors in the host cell despite the intrinsically unstable nature of F-box proteins, however, remains unclear. We found that the Agrobacterium F-box protein VirF, an important virulence factor, undergoes rapid degradation through the host proteasome pathway. This destabilization of VirF was counteracted by VirD5, another bacterial effector that physically associated with VirF. These observations reveal a previously unknown counterdefense strategy used by pathogens against potential host antimicrobial responses.