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.

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.

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.

Nuclear import of the capsid protein of tomato yellow leaf curl virus (TYLCV) in plant and insect cells.

The tomato yellow leaf curl virus (TYLCV) found in Israel is a whitefly-transmitted monopartite geminivirus. Although geminiviruses have been found in the nuclei of phloem-associated cells, the mechanism of viral invasion is poorly understood. The possible role of the TYLCV capsid protein (CP), the only known component of the viral coat, in virus transport into the host cell nucleus was investigated by monitoring its specific nuclear accumulation in plant and insect cells. CP was fused to the beta-glucuronidase (GUS) reporter enzyme to assay nuclear import in petunia protoplasts, and micro-injection of purified fluorescently labeled CP was used to examine its nuclear uptake in Drosophila embryos. Both assays demonstrated that TYLCV CP is transported into plant- and insect-cell nuclei by an active process of nuclear import via a nuclear localization signal (NLS)-specific pathway. Using the GUS assay and deletion analysis, the TYLCV CP NLS sequence was identified in the amino-terminus of the protein.

Transgenic tomato plants expressing the tomato yellow leaf curl virus capsid protein are resistant to the virus.

The tomato yellow leaf curl virus (TYLCV) gene that encodes the capsid protein (V1) was placed under transcriptional control of the cauliflower mosaic virus 35S promoter and cloned into an Agrobacterium Ti-derived plasmid and used to transform plants from an interspecific tomato hybrid, Lycopersicon esculentum X L. pennellii (F1), sensitive to the TYLCV disease. When transgenic F1 plants, expressing the V1 gene, were inoculated with TYLCV using whiteflies fed on TYLCV-infected plants, they responded either as untransformed tomato or showed expression of delayed disease symptoms and recovery from the disease with increasingly more resistance upon repeated inoculation. Transformed plants that were as sensitive to inoculation as untransformed controls expressed the V1 gene at the RNA level only. All the transformed plants that recovered from disease expressed the TYLCV capsid protein.