The Agrobacterium single-stranded DNA (ssDNA) intermediate T-strand is likely transferred to the plant cell nucleus as a complex with a single VirD2 molecule at its 5' end and multiple VirE2 molecules along its length. VirD2 contains a nuclear localization signal (NLS); however, because the T-strand is principally coated with VirE2 molecules, VirE2 also might assist in nuclear uptake. Indeed, VirE2 fused to a reporter protein localizes to plant cell nuclei, a process mediated by two amino acid sequences with homology to the bipartite NLS of Xenopus nucleoplasmin. Moreover, tumorigenicity of an avirulent virE2 mutant is restored when inoculated on transgenic plants expressing VirE2, supporting in planta function of VirE2.

This review is dedicated to Jeff Schell, one of the founders of modern`Agrobiology', the genetic and molecular dissection of crown gall disease. Together with notable scientists at the University of Gent, Belgium, Jeff spearheaded the discovery of the Ti-plasmid. The elusive`tumor inducing principle' was uncloaked and provided impetus for an incredibly fruitful subsequent 25 years of analyses. Scientists all over the world were caught up in unraveling the underlying mechanisms of Agrobacterium-mediated gene transfer to plants, and along the way uncovered a movable feast of fundamental insights. Below we summarize a sampling of Agrobacterium's most recently recognized accomplishments.

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Numerous bacterial pathogens use type IV secretion systems (T4SS) to deliver virulence factors directly to the cytoplasm of plant, animal, and human host cells. Here, evidence for interactions among components of the Agrobacterium tumefaciens virencoded T4SS is presented. The results derive from a high-resolution yeast two-hybrid assay, in which a library of small peptide domains of T4SS components was screened for interactions. The use of small peptides overcomes problems associated with assaying for interactions involving membrane-associated proteins. We established interactions between VirB11 (an inner membrane poreforming protein), VirB9 (a periplasmic protein), and VirB7 (an outer membrane-associated lipoprotein and putative pilus component). We provide evidence for an interaction pathway, among conserved members of a T4SS, spanning the A. tumefaciens envelope and including a potential pore protein. In addition, we have determined interactions between VirB1 (a lytic transglycosylase likely involved in the local remodeling of the peptidoglycan) and primarily VirB8, but also VirB4, VirB10, and VirB11 (proteins likely to assemble the core structure of the T4SS). VirB4 interacts with VirB8, VirB10, and VirB11, also establishing a connection to the core components. The identification of these interactions suggests a model for assembly of the T4SS.

Tobacco mosaic virus movement protein P30 complexes with genomic viral RNA for transport through plasmodesmata, the plant intercellular connections. Although most research with P30 focuses on its targeting to and gating of plasmodesmata, the mechanisms of P30 intracellular movement to plasmodesmata have not been defined. To examine P30 intracellular localization, we used tobacco protoplasts, which lack plasmodesmata, for transfection with plasmids carrying P30 coding sequences under a constitutive promoter and for infection with tobacco mosaic virus particles. In both systems, P30 appears as filaments that colocalize primarily with microtubules. To a lesser extent, P30 filaments colocalize with actin filaments, and in vitro experiments suggested that P30 can bind directly to actin and tubulin. This association of P30 with cytoskeletal elements may play a critical role in intracellular transport of the P30-viral RNA complex through the cytoplasm to and possibly through plasmodesmata.