Mechanism of activation of Agrobacterium virulence genes: identification of phenol-binding proteins.

Abstract

Agrobacterium tumefaciens initiates the expression of pathogenic genes (vir genes) in response to hostderived phenolic signals through a two-component regulatory system consisting of VirA and VirG. a-Bromoacetosyringone (ASBr) was developed as an inhibitor of this induction process and found to be a specific and irreversible inhibitor of vir gene induction in this pathogen. Formal replacement of one of the methoxy groups of ASBr with Iodine gave an equally effective inhibitor that could carry an 'mI label. We report here that the resulting radiolabeled inhibitor does not react with the sensory component of this system, VirA, either in vivo or in vitro. Rather, two small proteins, p1O and p2l, bind labeled inhibitor in vivo in a time period that is consistent with the exposure time required for the inhibition of vir gene expression. Labeling to these proteins was protected by preexposure to ASBr but not by a-bromo-3,5-dlmethoxyacetophenone, a compound of comparable chemical reactivity but previously shown not to inhibit vir gene expression. Our findings suggest that proteins that are not tumor-inducing plasmid-encoded mediate vir gene activation in a step prior to the VirA/VirG two-component regulatory system. Agrobacterium tumefaciens is the causative agent of crown gall tumors, a neoplastic disease of many dicotyledonous plants (1,2). A small oncogenic segment [transferred DNA (T-DNA)] of the tumor-inducing (Ti) plasmid harbored by these bacteria is transferred to the host cell, inducing the disease state. Many ofthe genes of the virulence regulon (vir) of the Ti plasmid required for this transfer are only expressed in the presence of host-derived phenolic compounds (3), one of which has been implicated in the control of division in the host cell (4-6).Sequence homology of two constitutively expressed genes of the vir regulon, virA and virG, with what now appears to be a large family of procaryotic two-component regulatory proteins (7) led to the proposal that these proteins served as the sensor/response elements controlling vir expression (8).This model predicts that VirA would interact with the phenolic inducer as the initiation step. Autophosphorylation in the C-terminal cytoplasmic domain of VirA (9, 10) and subsequent phosphate transfer to the transcriptional regulator, VirG (11), have been demonstrated in vitro and together constitute a phenol-activated signaling pathway. At least two other environmental stimuli, acidic pH and monosaccharides, act synergistically with the phenolic signals to activate vir gene expression (2).The dissection of the structures of these phenolic inducers has provided sufficient insight for the development of specific, irreversible inhibitors of vir gene expression (12). We now provide evidence that these inhibitors do not interact directly with VirA but rather with two other proteins, p10 and p21. We propose that these chromosomally encoded proteins modulate the signal transduction pathway prior to the autophosphorylation of VirA. MATERIALS AND METHODS Syntheses. a-Bro...

Agrobacterium-Mediated Plant Transformation: Biology and Applications

“…To initiate the signaling pathway, plant phenolics interact either directly or indirectly with the transmembrane sensory protein VirA. Although two chromosomally encoded proteins, p10 and p21, but not VirA, are able to bind the phenolic signal in vitro (Lee et al, 1992), genetic evidence strongly suggests that VirA directly senses the phenolic compounds required for vir gene activation (Lee et al, 1995;Lee et al, 1996). ChvE, a chromosomally encoded glucose/galactose binding protein, interacts with VirA and enhances vir gene activation by binding to sugars (Shimoda et al, 1993;Doty et al, 1996).…”

Phenolic acids act as signaling molecules in plant-microbe symbioses

“…Vir genes are induced at an acidic pH by phenolic compounds that function in concert with monosaccharides synthesized and exuded from wounded plant cells. Lee et al 122,123 clearly stated that the virA locus determines which phenolic compounds can function as vir gene inducers and the VirA protein directly senses the phenolic compounds for vir gene activation.…”

Detection of and Response to Signals Involved in Host-Microbe Interactions by Plant-Associated Bacteria

“…In one study, a radiolabeled phenolic compound failed to bind VirA but instead bound two small proteins (281). However, genetic evidence suggests that VirA is the direct receptor.…”