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.

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In host plants, cell-to-cell spread of tobacco mosaic virus (TMV) presumably occurs through intercellular connections, the plasmodesmata. TMV movement is mediated by a specific virus-encoded single-strand nucleic acid-binding protein, P30. The mechanism by which P30 operates is largely unknown. Here, we demonstrate that P30 expressed in transgenic plants is a phosphoprotein. We have developed an assay for in vitro phosphorylation of purified P30 by plant cell wall fractions and have localized the phosphorylation sites to amino acid residues Ser-258, Thr-261, and Ser-265. Interestingly, the P30 phosphorylation sites do not correspond to any known consensus phosphorylation sites for protein kinases. While P30 binding to single-stranded DNA (ssDNA) was shown to involve Thr-261, phosphorylation of this residue does not appear to play a role in binding activity. The protein kinase activity contained in the cell wall fractions was developmentally regulated, expressed predominantly in leaves. Within a leaf, this protein kinase activity increased with leaf maturation and correlated with the reported development of secondary plasmodesmata, sites of P30 accumulation. We suggest that phosphorylation may represent a mechanism for the host plant to sequester P30 following its localization to cell walls.[Key Words: TMV movement; plasmodesmata; protein kinase; phosphorylation; protein-nucleic acid binding.] In plants, viruses presumably spread from infected to adjacent healthy cells through intercellular connections, the plasmodesmata. Cell-to-cell movement of plant viruses is an active process mediated by specific virus-encoded movement proteins [for review, see Atabekov and Taliansky 1990;Citovsky and Zambryski 1991;Maule 1991;Deom et al. 1992). The best characterized movement protein is the P30 protein of tobacco mosaic virus (TMV). Three biological activities have been attributed to P30: (1) localization to plasmodesmata (Tomenius et al. 1987;Ding et al. 1992}; (2} increase in plasmodesmal permeability (Wolf et al. 1989}; and (3) cooperative binding to single-strand nucleic acids (Citovsky et al. 1990. On the basis of these observations, P30 was proposed to form complexes with the transported genomic TMV RNA and to target these complexes to and through the enlarged plasmodesmata channels (Citovsky and Zambryski 1991).The molecular mechanism by which P30 operates is still obscure. By analogy with many biological macromolecules, one possibility for control of P30 activity is via post-translational modification, such as phosphorylation. P30 was shown to be phosphorylated when ex- pressed from a baculovirus vector in insect cells (Atkins et al. 1991). However, the potential ability of host plants to phosphorylate P30 has not been demonstrated directly nor have the P30 phosphorylation sites been identified. Furthermore, the effect of P30 phosphorylation on the biological activity of this protein is unknown.Here, we demonstrate that P30 is phosphorylated at carboxy-terminal serine (S-258 and S-265) and threonine {Thr-261) resid...

The synthesis of peptidoglycan (PG) in bacteria is a crucial process controlling cell shape and vitality. In contrast to bacteria such as Escherichia coli that grow by dispersed lateral insertion of PG, little is known of the processes that direct polar PG synthesis in other bacteria such as the Rhizobiales. To better understand polar growth in the Rhizobiales Agrobacterium tumefaciens, we first surveyed its genome to identify homologs of (~70) well-known PG synthesis components. Since most of the canonical cell elongation components are absent from A. tumefaciens, we made fluorescent protein fusions to other putative PG synthesis components to assay their subcellular localization patterns. The cell division scaffolds FtsZ and FtsA, PBP1a, and a Rhizobiales- and Rhodobacterales-specific l,d-transpeptidase (LDT) all associate with the elongating cell pole. All four proteins also localize to the septum during cell division. Examination of the dimensions of growing cells revealed that new cell compartments gradually increase in width as they grow in length. This increase in cell width is coincident with an expanded region of LDT-mediated PG synthesis activity, as measured directly through incorporation of exogenous d-amino acids. Thus, unipolar growth in the Rhizobiales is surprisingly dynamic and represents a significant departure from the canonical growth mechanism of E. coli and other well-studied bacilli.