ChvE is a chromosomally encoded protein inThe expression of vir genes in A. tumefaciens is activated by plant-released signals, namely, phenolic derivatives, acidic pH, and monosaccharides (for a review, see reference 6), via the combined activities of the periplasmic protein ChvE and the VirA/VirG two-component regulatory system. Upon perception of these plant signals, autophosphorylated VirA, a transmembrane histidine kinase, transfers a phosphoryl group to VirG, a response regulator, and then the phosphorylated VirG activates the expression of vir genes by binding vir boxes in their promoters (8,19,24,31,52).Perception and transduction of the sugar signals is crucial to the virulence of A. tumefaciens: strains lacking ChvE, a chromosomally encoded putative sugar-binding protein, are significantly less virulent than wild-type strains (17,18). Previous studies have shown that, in fact, sugar signaling is neither sufficient for nor absolutely required for vir gene expression. Rather, sugars vastly increase both the sensitivity of VirA to phenol derivatives, such as acetosyringone (AS), and the maximal levels of vir gene expression observed at saturating levels of such compounds (for a review, see reference 26). The periplasmic domain of VirA is required for transduction of the sugar and pH signals (7,8,16,41), whereas the so-called "linker" domain, located in the cytoplasm between the second transmembrane domain and the kinase domain, is required for perception and transduction of the phenolic signals (8,46,47).A working model for the ChvE/sugar/VirA signaling pathway suggests that monosaccharide-bound ChvE interacts with the periplasmic domain of VirA to relieve periplasmic repression, resulting in maximal sensitivity of VirA to phenolic signals (7,11,32,41). However, limited evidence has been presented to reveal how ChvE recognizes monosaccharides and how it interacts with the periplasmic domain of VirA. Shimoda et al. (41) identified a mutant chvE allele [chvE(T211M)] that is able to suppress a sugar-insensitive virA allele [virA(E210V)], thereby restoring the sugar-sensing ability. The suppressing effect of chvE(T211M) was then proposed to be the result of the specific restoration of the capacity of VirA E210V to bind ChvE T211M . However, ChvE T211M also activated wild-type VirA in the absence of sugars (32), suggesting that this mutant may not be a site-specific suppressor of VirA E210V . Based on a homology model of ChvE, a recent study (16) does predict, though, that the residue T211 is located on the surface of the

Isogenic strains of Agrobacterium tumefaciens carrying pTiC58, pAtC58, or both were constructed and assayed semiquantitatively and quantitatively for virulence and vir gene expression to study the effect of the large 542-kb accessory plasmid, pAtC58, on virulence. Earlier studies indicate that the att (attachment) genes of A. tumefaciens are crucial in the ability of this soil phytopathogen to infect susceptible host plants. Mutations in many att genes, notably attR and attD, rendered the strain avirulent. These genes are located on pAtC58. Previous work also has shown that derivatives of the wild-type strain C58 cured of pAtC58 are virulent as determined by qualitative virulence assays and, hence, pAtC58 was described as nonessential for virulence. We show here that the absence of pAtC58 in pTiC58-containing strains results in reduced virulence but that disruption of the attR gene does not result in avirulence or a reduction in virulence. Our studies indicate that pAtC58 has a positive effect on vir gene induction as revealed by immunoblot analysis of Vir proteins and expression of a P virB ::lacZ fusion.Agrobacterium tumefaciens is a soil phytopathogen that incites tumors on susceptible plants by transferring the T-DNA, a portion of its tumor-inducing plasmid (pTi), into plant cells (Van Larebeke et al., 1974; Zhu et al., 2000; Zupan et al., 2000; Gelvin, 2003). This T-DNA is translocated to the nucleus, integrated into plant chromosomal DNA, and expressed. In strain C58, the products of approximately 20 vir genes arranged in six operons (virA, virB, virC, virD, virE, and virG) on pTiC58, as well as several chromosomal genes, act in a concerted fashion to induce tumor formation on host plants. Two critical early steps in this process are the attachment of the bacterium to the plant cell and the activation of virulence genes. Subsequently, a DNA-protein intermediate is formed and crosses the bacterium-plant interface, followed by the transport of this intermediate into the plant cell nucleus, integration of the T-DNA into the plant chromosome and expression of the T-DNA (Zupan et al., 2000; Christie, 2001; Tzfira and Citovsky, 2002; Gelvin, 2003). In nature, this results in the formation of continuously proliferating tumors that produce novel metabolites (opines) that can be utilized by the inciting bacterium but not by the plant (Dessaux et al., 1998).Chromosomal and Ti plasmid genes are involved in the recognition of a plant environment and activation of the virulence machinery. The Ti-encoded VirA/VirG two-component regulatory system activates vir gene expression in response to the integrated effects of phenols, sugars, and low pH that are found at the plant wound site (Stachel and Zambryski, 1986;Shimoda et al., 1990; Turk et al., 1991; Winans, 1991; Winans et al., 1994; Lee et al., 1995). chvE is a chromosomal virulence gene encoding a periplasmic sugar-binding protein that interacts with the periplasmic domain of VirA, resulting in optimal expression of vir genes (Cangelosi et al., 1990; Huang et a...

The plant pathogen Agrobacterium tumefaciens responds to three main signals at the plant-bacterium interface: phenolics, such as acetosyringone (AS), monosaccharides, and acidic pH (ϳ5.5). These signals are transduced via the chromosomally encoded sugar binding protein ChvE and the Ti plasmid-encoded VirA/VirG two-component regulatory system, resulting in the transcriptional activation of the Ti plasmid virulence genes. Here, we present genetic and physical evidence that the periplasmic domain of VirA dimerizes independently of other parts of the protein, and we examine the effects of several engineered mutations in the periplasmic and transmembrane regions of VirA on vir-inducing capacity as indicated by AS sensitivity and maximal level of vir-inducing activity at saturating AS levels. The data indicate that helix-breaking mutations throughout the periplasmic domain of VirA or mutations that reposition the second transmembrane domain (TM2) of VirA relieve the periplasmic domain's repressive effects on the maximal activity of this kinase in response to phenolics, effects normally relieved only when ChvE, sugars, and low pH are also present. Such relief, however, does not sensitize VirA to low concentrations of phenolics, the other major effect of the ChvE-sugar and low pH signals. We further demonstrate that amino acid residues in a small Trg-like motif in the periplasmic domain of VirA are crucial for transmission of the ChvE-sugar signal to the cytoplasmic domain. These experiments provide evidence that small perturbations in the periplasmic domain of VirA can uncouple sugar-mediated changes in AS sensitivity from the sugar-mediated effects on maximal activity.