Biomedical subjects

Yedidya Gafni

Publications and source records attributed to Yedidya Gafni.

9 recordsLinked to original sources

PubMed2006

Production of siRNA targeted against TYLCV coat protein transcripts leads to silencing of its expression and resistance to the virus.

The coat protein (CP) of Tomato yellow leaf curl virus (TYLCV), encoded by the v1 gene, is the only known component of the viral capsid. In addition, the CP plays a role in the virus transport into the host cell nucleus where viral genes are replicated and transcribed. In this study, we analyzed the effect of small interfering double-stranded RNAs (siRNAs), derived from an intron-hairpin RNA (ihpRNA) construct and targeting the v1 gene product, on CP accumulation. Transient assays involving agroinfiltration of the CP-silencing construct followed by infiltration of a fused GFP-CP (green fluorescent protein-coat protein) gene showed down-regulation of GFP expression in Nicotiana benthamiana. Some of the transgenic tomato plants (cv. Micro-Tom), expressing the siRNA targeted against the TYLCV CP gene, did not show disease symptoms 7 weeks post-inoculation with the virus, while non-transgenic control plants were infected within 2 weeks post inoculation. The present study demonstrates, for the first time, that siRNA targeted against the CP of TYLCV can confer resistance to the virus in transgenic tomato plants, thereby enabling flowering and fruit production.

Agrobacterium tumefaciens↗

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

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

Permeabilized mammalian cells as an experimental system for nuclear import of geminiviral karyophilic proteins and of synthetic peptides derived from their nuclear localization signal regions.

The plant-infecting geminiviruses deliver their genome and viral proteins into the host cell nucleus. Members of the family Geminiviridae possess either a bipartite genome composed of two approximately 2.6 kb DNAs or a monopartite genome of approximately 3.0 kb DNA. The bipartite genome of Bean dwarf mosaic virus (BDMV) encodes several karyophilic proteins, among them the capsid protein (CP) and BV1 (nuclear shuttle protein). A CP is also encoded by the monopartite genome of Tomato yellow leaf curl virus (TYLCV). Here, an in vitro assay system was used for direct demonstration of nuclear import of BDMV BV1 and TYLCV CP, as well as synthetic peptides containing their putative nuclear localization signals (NLSs). Full-length recombinant BDMV BV1 and TYLCV CP mediated import of conjugated fluorescently labelled BSA molecules into nuclei of permeabilized mammalian cells. Fluorescently labelled and biotinylated BSA conjugates bearing the synthetic peptides containing aa 3-20 of TYLCV CP (CP-NLS) or aa 84-106 of BDMV BV1 (BV1-NLS) were also imported into the nuclei of permeabilized cells. This import was blocked by the addition of unlabelled BSA-NLS peptide conjugates or excess unlabelled free NLS peptides. The CP- and BV1-NLS peptides also mediated nuclear import of fluorescently labelled BSA molecules into the nuclei of microinjected mesophyll cells of Nicotiana benthamiana leaves, demonstrating their biological function in intact plant tissue. BV1-NLS and CP-NLS were shown to mediate specific binding to importin alpha, both in vitro and in vivo. These results are consistent with a common nuclear-import pathway for CP and BV1, probably via importin alpha.

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

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

Coniferyl alcohol, a lignin precursor, stimulates Rhizobium rhizogenes A4 virulence.

Rhizobium rhizogenes, a soil bacterium, is the causative agent of the neoplastic disease hairy root. Upon incubation of Rhizobium rhizogenes A4 with coniferyl alcohol, a lignin precursor, bacterial virulence on cotton cotyledon slices was stimulated. This was observed both in numbers of root hairs produced and in their length. Stimulation was maximized after exposure of bacteria to 150 microg/mL of coniferyl alcohol for 4 h. This was shown to be at the early log phase of bacterial growth.

Culture Media↗

PubMed2004

Non-endocytic penetration of core histones into petunia protoplasts and cultured cells: a novel mechanism for the introduction of macromolecules into plant cells.

The results of the present work demonstrate that core histones are able to penetrate the plasma membrane of plant cells. Confocal microscopy has revealed that incubation of petunia protoplasts with fluorescently labeled core histones resulted in cell penetration and nuclear import of the externally added histones. Intracellular accumulation was also confirmed by an ELISA-based quantitative method using biotin-labeled histones. Penetration into petunia protoplasts and cultured cells was found to be non-saturable, occurred at room temperature and at 4 degrees C and was not inhibited by Nocodazole. Furthermore, penetration of the biotinylated histone was neither blocked by the addition of an excess of free biotin molecules, nor by non-biotinylated histone molecules. All these results clearly indicate that the observed uptake is due to direct translocation through the cell plasma membrane and does not occur via endocytosis. Our results also show that the histones H2A and H4 were able to mediate penetration of covalently attached BSA molecules demonstrating the potential of the histones as carriers for the delivery of macromolecules into plant cells. To the best of our knowledge, the findings of the present paper demonstrate, for the first time, the activity of cell penetrating proteins (CPPs) in plant cells.

Cells, Cultured↗

PubMed2004

Cloning and characterization of the tomato karyopherin alpha1 gene promoter.

The karyopherin alpha1 (LeKAPalpha 1) gene of tomato (Lycopersicon esculentum) encodes a receptor involved in nuclear import. To analyze the expression pattern of this gene, a genomic clone containing its upstream region was isolated and sequenced. To study the promoter functionality, a 2170 bp fragment (LM1), was fused to glucuronidase (GUS) and introduced into petunia cells by particle bombardment. For further characterization of the promoter, one inverse and three deletion constructs were studied in cell suspension. To follow its expression in tobacco leaves, transgenic plants expressing GUS under the control of the LM1 promoter were made. Expression of LM1-GUS was largely restricted to actively growing leaf regions, suggesting possible involvement of active cell division and plant growth regulators in LeKAPalpha 1 expression.

Base Sequence↗