Two African swine fever virus proteins derived from a common precursor exhibit different nucleocytoplasmic transport activities.

African swine fever virus (ASFV), a large icosahedral deoxyvirus, is the causative agent of an economically relevant hemorrhagic disease that affects domestic pigs. The major purpose of the present study was to investigate the nuclear transport activities of the ASFV p37 and p14 proteins, which result from the proteolytic processing of a common precursor. Experiments were performed by using yeast-based nucleocytoplasmic transport assays and by analysis of the subcellular localization of different green fluorescent and Myc fusion proteins in mammalian cells. The results obtained both in yeast and mammalian cells clearly demonstrated that ASFV p14 protein is imported into the nucleus but not exported to the cytoplasm. The ability of p37 protein to be exported from the nucleus to the cytoplasm of both yeast and mammalian cells was also demonstrated, and the results clearly indicate that p37 nuclear export is dependent on the interaction of the protein with the CRM-1 receptor. In addition, p37 was shown to exhibit nuclear import activity in mammalian cells. The p37 protein nuclear import and export abilities described here constitute the first report of a nucleocytoplasmic shuttling protein encoded by the ASFV genome. Overall, the overlapping results obtained for green fluorescent protein fusions and Myc-tagged proteins undoubtedly demonstrate that ASFV p37 and p14 proteins exhibit nucleocytoplasmic transport activities. These findings are significant for understanding the role these proteins play in the replication cycle of ASFV.

RNA commutes to work: regulation of plant gene expression by systemically transported RNA molecules.

Although long-distance movement of endogenous mRNAs in plants is well established, the functional contributions of these transported RNA molecules has remained unclear. In a recent report, Kim et al.2001 showed that systemically transported mRNA is capable of causing phenotypic change in developing tissue. Here, this finding and its significance are reviewed and discussed in detail. In addition, in order to give proper perspective, long-distance transport of other types of RNAs, e.g., RNA elicitors of post-transcriptional gene silencing and RNA genomes of plant viruses, and its possible regulation are discussed.

Inhibition of systemic onset of post-transcriptional gene silencing by non-toxic concentrations of cadmium.

Post-transcriptional gene silencing (PTGS) is an important mechanism for regulation of plant gene expression and virus-plant interactions. To better understand this process, the heavy metal cadmium was identified as a specific inhibitor in two different PTGS systems, constitutive and inducible. The pattern of cadmium-induced inhibition of PTGS allowed several insights into PTGS development. First, cadmium treatment prevented only systemic but not local onset of PTGS, uncoupling between these two modes of PTGS. Second, non-toxic, but not toxic, levels of cadmium inhibited PTGS, suggesting induction of a pathway that interferes with PTGS. Third, cadmium effects on PTGS closely paralleled those on the movement of tobamoviruses, suggesting that both processes may share common steps in their systemic transport pathways. Interestingly, these effects of cadmium do not represent a general property of toxic metal ions because two other such elements, that is zinc and aluminum, did not interfere with PTGS and viral systemic movement.

VIP1, an Arabidopsis protein that interacts with Agrobacterium VirE2, is involved in VirE2 nuclear import and Agrobacterium infectivity.

T-DNA nuclear import is a central event in genetic transformation of plant cells by Agrobacterium. This event is thought to be mediated by two bacterial proteins, VirD2 and VirE2, which are associated with the transported T-DNA molecule. While VirD2 is imported into the nuclei of plant, animal and yeast cells, nuclear uptake of VirE2 occurs most efficiently in plant cells. To understand better the mechanism of VirE2 action, a cellular interactor of VirE2 was identified and its encoding gene cloned from Arabidopsis. The identified plant protein, designated VIP1, specifically bound VirE2 and allowed its nuclear import in non-plant systems. In plants, VIP1 was required for VirE2 nuclear import and Agrobacterium tumorigenicity, participating in early stages of T-DNA expression.

Genetic transformation of HeLa cells by Agrobacterium.

Agrobacterium tumefaciens is a soil phytopathogen that elicits neoplastic growths on the host plant species. In nature, however, Agrobacterium also may encounter organisms belonging to other kingdoms such as insects and animals that feed on the infected plants. Can Agrobacterium, then, also infect animal cells? Here, we report that Agrobacterium attaches to and genetically transforms several types of human cells. In stably transformed HeLa cells, the integration event occurred at the right border of the tumor-inducing plasmid's transferred-DNA (T-DNA), suggesting bona fide T-DNA transfer and lending support to the notion that Agrobacterium transforms human cells by a mechanism similar to that which it uses for transformation of plants cells. Collectively, our results suggest that Agrobacterium can transport its T-DNA to human cells and integrate it into their genome.

Comparison between nuclear localization of nopaline- and octopine-specific Agrobacterium VirE2 proteins in plant, yeast and mammalian cells.

SUMMARY In a unique case of trans-kingdom DNA transfer, Agrobacterium genetically transforms plants by transferring its DNA segment into the host cell nucleus and integrating it into the plant genome. One of the central players in this process is the bacterial virulence protein, VirE2, which binds the transported DNA molecule and facilitates its nuclear import. Nuclear import of VirE2 proteins encoded by two major Agrobacterium strains, nopaline and octopine, has been hypothesized to occur by different mechanisms, i.e. the nopaline VirE2 was imported only into the nuclei of plant cells while the octopine VirE2 also accumulated in the nuclei of animal cells. Here, this notion was tested by a systematic comparison of nuclear import of nopaline- and octopine-specific VirE2 in dicotyledonous and monocotyledonous plants and in living mammalian and yeast cells. These experiments showed that nuclear import of both nopaline and octopine VirE2 proteins is plant-specific, occurring in plant but not in non-plant systems.

Nucleic acid transport in plant-microbe interactions: the molecules that walk through the walls.

Many microbes "genetically invade" plants by introducing DNA or RNA molecules into the host cells. For example, plant viruses transport their genomes between host cells, whereas Agrobacterium spp. transfer T-DNA to the cell nucleus and integrate it into the plant DNA. During these events, the transported nucleic acids must negotiate several barriers, such as plant cell walls, plasma membranes, and nuclear envelopes. This review describes the microbial and host proteins that participate in cell-to-cell transport and nuclear import of nucleic acids during infection by plant viruses and Agrobacterium spp. Possible molecular mechanisms by which these transport processes occur are discussed.

Regulation of plasmodesmal transport by phosphorylation of tobacco mosaic virus cell-to-cell movement protein.

Cell-to-cell spread of tobacco mosaic virus (TMV) through plant intercellular connections, the plasmodesmata, is mediated by a specialized viral movement protein (MP). In vivo studies using transgenic tobacco plants showed that MP is phosphorylated at its C-terminus at amino acid residues Ser258, Thr261 and Ser265. When MP phosphorylation was mimicked by negatively charged amino acid substitutions, MP lost its ability to gate plasmodesmata. This effect on MP-plasmodesmata interactions was specific because other activities of MP, such as RNA binding and interaction with pectin methylesterases, were not affected. Furthermore, TMV encoding the MP mutant mimicking phosphorylation was unable to spread from cell to cell in inoculated tobacco plants. The regulatory effect of MP phosphorylation on plasmodesmal permeability was host dependent, occurring in tobacco but not in a more promiscuous Nicotiana benthamiana host. Thus, phosphorylation may represent a regulatory mechanism for controlling the TMV MP-plasmodesmata interactions in a host-dependent fashion.

A genetic system for detection of protein nuclear import and export.

We have developed a simple genetic assay to detect active nuclear localization (NLS) and export signals (NES) on the basis of their function within yeast cells. The bacterial LexA protein was modified (mLexA) to abolish its intrinsic NLS and fused to the activation domain of the yeast Gal4p (Gal4AD) with or without the SV40 large T-antigen NLS. In the import assay, if a tested protein fused to mLexA-Gal4AD contains a functional NLS, it will enter the cell nucleus and activate the reporter gene expression. In the export assay, if a tested protein fused to mLexA-SV40 NLS-Gal4AD contains a functional NES, it will exit into the cytoplasm, decreasing the reporter gene expression. We tested this system with known NLS and NES and then used it to demonstrate a NES activity of the capsid protein of a plant geminivirus. This approach may help to identify, analyze, and select for proteins containing functional NLS and NES.

Interaction between the tobacco mosaic virus movement protein and host cell pectin methylesterases is required for viral cell-to-cell movement.

Virus-encoded movement protein (MP) mediates cell-to-cell spread of tobacco mosaic virus (TMV) through plant intercellular connections, the plasmodesmata. The molecular pathway by which TMV MP interacts with the host cell is largely unknown. To understand this process better, a cell wall-associated protein that specifically binds the viral MP was purified from tobacco leaf cell walls and identified as pectin methylesterase (PME). In addition to TMV MP, PME is recognized by MPs of turnip vein clearing virus (TVCV) and cauliflower mosaic virus (CaMV). The use of amino acid deletion mutants of TMV MP showed that its domain was necessary and sufficient for association with PME. Deletion of the PME-binding region resulted in inactivation of TMV cell-to-cell movement.

Cell-to-cell movement of tobacco mosaic virus: enigmas and explanations.

Abstract Tobacco mosaic virus (TMV) spreads between cells through plant intercellular connections, the plasmodesmata. This transport process is mediated by a specialized virus-encoded movement protein, TMV MP. Recent advances in two major aspects of TMV MP function highlight the limits of our current knowledge and promise exciting future developments. First, findings that TMV MP interacts with cytoskeletal elements and cell wall proteins suggest potential mechanisms for TMV MP targeting from the cell cytoplasm to plasmodesmal channels. Second, indications that TMV MP phosphorylation plays a regulatory role in several activities of TMV MP begin to unravel molecular pathways that control TMV cell-to-cell transport. TMV systemic movement that follows its initial cell-to-cell spread, on the other hand, may be controlled through two different pathways used for viral entry into and exit from the host plant vascular tissue.

From host recognition to T-DNA integration: the function of bacterial and plant genes in the Agrobacterium-plant cell interaction.

UNLABELLED: Abstract Agrobacterium tumefaciens and its related species, A. rhizogenes and A. vitis, are the only known bacterial pathogens which 'genetically invade' host plants and stably integrate part of their genetic material into the host cell genome. Thus, A. tumefaciens has evolved as a major tool for plant genetic engineering. Furthermore, this unique process of interkingdom DNA transfer has been utilized as a model system for studies of its underlying biological events, such as intercellular signalling, cell-to-cell DNA transport, protein and DNA nuclear import and integration. To date, numerous bacterial proteins and several plant proteins have been implicated in the A. tumefaciens-plant cell interaction. Here, we discuss the molecular interactions among these bacterial and plant factors and their role in the A. tumefaciens-plant cell DNA transfer. Taxonomic relationship: Bacteria; Proteobacteria; alpha subdivision; Rhizobiaceae group; Rhizobiaceae family; Agrobacterium genus. Microbiological properties: Gram-negative, nonsporing, motile, rod-shaped, soil-borne. Related species:A. rhizogenes (causes root formation in infected plants), A. vitis (causes gall formation on grapevines). Disease symptoms: Formation of neoplastic swellings (galls) on plant roots, crowns, trunks and canes. Galls interfere with water and nutrient flow in the plants, and seriously infected plants suffer from weak, stunted growth and low productivity. HOST RANGE: One of the widest host ranges known among plant pathogens; can potentially attack all dicotyledonous plant species. Also, under controlled conditions (usually in tissue culture), can infect, albeit with lower efficiency, several monocotyledonous species. Agronomic importance: The disease currently affects plants belonging to the rose family, e.g. apple, pear, peach, cherry, almond, roses, as well as poplar trees (aspen). Useful web site:http://www.bio.purdue.edu/courses/gelvinweb/gelvin.html.

Tobacco mosaic virus: a pioneer of cell-to-cell movement.

Cell-to-cell movement of tobacco mosaic virus (TMV) is used to illustrate macromolecular traffic through plant intercellular connections, the plasmodesmata. This transport process is mediated by a specialized viral movement protein, P30. In the initially infected cell, P30 is produced by transcription of a subgenomic RNA derived from the invading virus. Presumably, P30 then associates with a certain proportion of the viral RNA molecules, sequestering them from replication and mediating their transport into neighbouring uninfected host cells. This nucleoprotem complex is targeted to plasmodesmata, possibly via interaction with the host cell's cytoskeleton. Prior to passage through a plasmodesma, the plasmodesmatal channel is dilated by the movement protein. It is proposed that targeting of P30-TMV RNA complexes to plasmodesmatal involves binding to a specific cell-wall-associated receptor molecule. This protein, designated p38, also functions as a protein kinase, phosphorylating P30 at its carboxy-terminus and minimizing P30-induced interference with plasmodesmatal permeability during viral infection.

Non-toxic concentrations of cadmium inhibit systemic movement of turnip vein clearing virus by a salicylic acid-independent mechanism.

Systemic movement of plant viruses is a central event in viral infection. To better understand this process, the heavy metal cadmium was used to inhibit systemic spread of turnip vein clearing virus (TVCV), a tobamovirus, in tobacco plants. Study of the mechanism by which cadmium exerts this inhibitory effect may provide insights into the essential steps of the TVCV systemic movement pathway. Our results demonstrated that cadmium treatment did not affect TVCV transport from the inoculated non-vascular tissue into the plant vasculature but blocked viral exit into uninoculated non-vascular tissues. Thus, TVCV virions may enter and exit the host plant vascular system by two different mechanisms. We also showed that cadmium-treated plants still supported systemic spread of an unrelated tobacco etch virus (TEV), suggesting multiple pathways for systemic infection. Finally, cadmium-induced arrest in TVCV systemic infection was shown to occur by a salicylic acid-independent mechanism.

The capsid protein of tomato yellow leaf curl virus binds cooperatively to single-stranded DNA.

The capsid protein (CP) of tomato yellow leaf curl virus (TYLCV) is the only known component of the virus coat. Here, we identify TYLCV CP as a single-stranded (ss) DNA binding protein. Purified TYLCV CP bound ssDNA in a highly cooperative and sequence-nonspecific fashion. TYLCV CP-ssDNA complexes were resistant to nucleolytic digestion and remained stable at relatively high salt concentrations. Because TYLCV CP is known to contain an active nuclear targeting signal, we propose that its association with the viral genomic ssDNA mediates TYLCV entry into the host cell nucleus during the infection process.

Preservation of plant cell ultrastructure during immunolocalization of virus particles.

Most immunoelectron microscopy techniques used for ultrastructural analyses of virus-infected plant tissues significantly compromise cellular membranous structures as well as overall contrast and resolution of the image. Here, we describe a protocol which avoids these flaws but retains full antigenicity of the sample. A direct comparison of the conventional and the improved electron immunostaining procedures is presented using tobacco and Arabidopsis thaliana plants infected with turnip vein clearing virus.

An Arabidopsis thaliana mutant with virus-inducible phenotype.

The role of host factors in plant viral diseases is not well understood. To study this important aspect of plant-pathogen interaction, we identified an Arabidopsis thaliana mutant, designated vid1 (virus-inducible dwarf), with altered responses to viral infection. Specifically, vid1 resembled the wild-type plants when healthy but developed a severely dwarfed phenotype with a loss of apical dominance following infection by a tobamovirus. Genetic segregation showed that the vid1 phenotype is caused by a recessive mutation in a single gene. Since systemic viral infection is thought to interfere with the host plant intercellular transport, we propose that the vid1 mutation affects this transport process. Combination of the mutation and viral infection may disrupt transport of developmental regulators, such as hormones, causing formation of the vid1 phenotype. Indeed, the effect of vid1 mutation was repressed by exogenous application of a plant hormone auxin. Potentially, the vid1 mutant will help characterize the mechanism of virus-plant interaction and formation of plant viral disease symptoms.

Identification of an Arabidopsis thaliana mutation (vsm1) that restricts systemic movement of tobamoviruses.

Following inoculation, many plant viruses spread locally from cell to cell until they reach the vascular system, through which they then move to other parts of the plant, resulting in systemic infection. To isolate host genes involved in systemic transport of plant viruses, ethyl methanesulfonate-mutagenized Arabidopsis thaliana plants were screened for significant delays in the systemic movement of turnip vein clearing virus (TCVC). One such mutant, designated vsm1 (virus systemic movement), was identified. Unlike the wild-type plants, vsm1 did not develop viral disease and did not allow the systemic spread of the virus. The local viral movement within the inoculated vsm1 leaves, however, was not affected. TVCV systemic movement within the vsm1 plants was likely blocked at the step of viral entry into the host plant vasculature from the infected leaf tissue. vsm1 plants also restricted the systemic movement of another tobamovirus but not of an unrelated carmovirus.