The initiation of intracellular infection of legume roots by symbiotic rhizobia bacteria and arbuscular mycorrhiza (AM) fungi is preceded by the induction of calcium signatures in and around the nucleus of root epidermal cells. Although a calcium and calmodulin-dependent kinase (CCaMK) is a key mediator of symbiotic root responses, the decoding of the calcium signal and the molecular events downstream are only poorly understood. Here, we characterize Lotus japonicus cyclops mutants on which microbial infection was severely inhibited. In contrast, nodule organogenesis was initiated in response to rhizobia, but arrested prematurely. This arrest was overcome when a deregulated CCaMK mutant version was introduced into cyclops mutants, conferring the development of full-sized, spontaneous nodules. Because cyclops mutants block symbiotic infection but are competent for nodule development, they reveal a bifurcation of signal transduction downstream of CCaMK. We identified CYCLOPS by positional cloning. CYCLOPS carries a functional nuclear localization signal and a predicted coiled-coil domain. We observed colocalization and physical interaction between CCaMK and CYCLOPS in plant and yeast cell nuclei in the absence of symbiotic stimulation. Importantly, CYCLOPS is a phosphorylation substrate of CCaMK in vitro. Cyclops mutants of rice were impaired in AM, and rice CYCLOPS could restore symbiosis in Lotus cyclops mutants, indicating a functional conservation across angiosperms. Our results suggest that CYCLOPS forms an ancient, preassembled signal transduction complex with CCaMK that is specifically required for infection, whereas organogenesis likely requires additional yet-to-be identified CCaMK interactors or substrates.BiFC ͉ map-based cloning ͉ plant-microbe symbiosis ͉ protein phosphorylation ͉ protein-protein interaction L egume plants can establish endosymbiotic interactions with nitrogen-fixing rhizobia and phosphate-delivering arbuscular mycorrhiza (AM) fungi. Plant root hairs form a tight curl in which rhizobia are entrapped. From this closed infection pocket, the bacteria are guided by plant membrane-delimited infection threads (ITs) into the root nodule, a specialized organ developed by the plant to provide an optimized environment for nitrogen fixation (1). AM fungal hyphae are guided through epidermal and cortical cells toward the inner cortex (2), where arbuscules, highly branched intracellular symbiotic structures, are formed (3). Intracellular infection by rhizobia and AM fungi is preceded by an exchange of specific signaling molecules. Rhizobia produce lipochito-oligosaccharides (Nod factors) that activate host plant responses including root hair deformation, and preinfection thread formation, which are structures that determine the path of IT growth through the root (4), and initiation of cortical cell division (1). One of the earliest plant responses to stimulation by Nod factors is Ca 2ϩ -spiking, which consists of perinuclear oscillations of calcium concentration in root cells (5). In the legume

The plant pathogen Agrobacterium tumefaciens induces the formation of crown gall tumours at wound sites on host plants by directly transforming plant cells. This disease strategy benefits the bacteria as the infected plant tissue produces novel nutrients, called opines, that the colonizing bacteria can use as nutrients. Almost all of the genes that are required for virulence, and all of the opine uptake and utilization genes, are carried on large tumour-inducing (Ti) plasmids. The observation more than 25 years ago that specific opines are required for Ti plasmid conjugal transfer led to the discovery of a cell-cell signalling system on these plasmids that is similar to the LuxR-LuxI system first described in Vibrio fischeri. All Ti plasmids that have been described to date carry a functional LuxI-type N-acylhomoserine lactone synthase (TraI), and a LuxR-type signal receptor and transcriptional regulator called TraR. The traR genes are expressed only in the presence of specific opines called conjugal opines. The TraR-TraI system provides an important model for LuxR-LuxI-type systems, especially those found in the agriculturally important Rhizobiaceae family. In this review, we discuss current advances in the biochemistry and structural biology of the TraR-TraI system.

SummaryBurkholderia cenocepacia is an opportunistic human pathogen that can aggressively colonize the cystic fibrosis lung. This organism has a LuxR/LuxI-type quorum sensing system that enables cell-cell communication via exchange of acyl homoserine lactones (AHLs). The CepR and CepI proteins constitute a global regulatory system, controlling expression of at least 40 genes, including those controlling swarming motility and biofilm formation. In this study, we isolated seven lacZ fusions in a clinical isolate of B. cenocepacia that are inducible by octanoyl-HSL. Induction of all of these genes requires CepR. The cepI promoter was tested for induction by a set of 33 synthetic autoinducers and analogues, and was most strongly induced by long-chain AHLs lacking 3-oxo substitutions. Expression of this promoter was inhibited by high concentrations of three different autoinducers, each having six-carbon acyl chains. When CepR protein was overproduced in Escherichia coli , it accumulated in a soluble form in the presence of octanoyl-HSL, but accumulated only as insoluble inclusion bodies in its absence. Purified CepR-OHL complexes bound to specific DNA sequences at the cepI and aidA promoters with high specificity. These binding sites included a 16-nucleotide imperfect dyad symmetry. Both CepR binding sites are centred approximately 44 nucleotides upstream of the respective transcription start sites.

SummaryTraR of Agrobacterium tumefaciens is a member of the LuxR family of transcriptional regulators, and binds to specific DNA sequences (tra boxes) at target promoters of the tumour-inducing (Ti) plasmid. Each tra box has a pronounced dyad symmetry, and each subunit of a TraR dimer binds to one half of a tra box via a helix-turn-helix (HTH) DNA binding motif. Structural analysis has suggested that TraR makes extensive sequence-specific contacts with tra box DNA. In this study, we tested these predictions using synthetic self-complementary oligonucleotides containing variant tra box sequences. Some predictions made from structural analysis were confirmed, while others were shown to be incorrect. Unexpectedly, these experiments also showed that six nucleotides at the centre of the tra box that make no direct contact with TraR are nevertheless critical for high-affinity binding and probably act by facilitating a previously described DNA bend. Variant tra boxes were also tested for transcription activity in vivo. Most transcription assays reflected in vitro binding assays. However, alterations of the outermost nucleotides had little effect on TraR binding but blocked transcription, probably by altering an overlapping -35 promoter motif.

SummaryThe LuxR-type quorum-sensing transcription factor TraR regulates replication and conjugal transfer of the tumour-inducing (Ti) plasmid in the plant pathogen Agrobacterium tumefaciens. TraR is a twodomain protein with an N-terminal domain that binds to the quorum-sensing signal N-3-oxooctanoyl-L -homoserine lactone (OOHL) and a C-terminal domain that binds to specific DNA sequences called tra boxes. TraR-OOHL complexes form homodimers that activate transcription of at least seven promoters on the Ti plasmid. At five promoters, a tra box overlaps the binding site of core RNA polymerase (class II promoters), while in the other two promoters, this site is located farther upstream (class I promoters). In this study, we performed saturating point mutagenesis of the surface residues of the TraR C-terminal domain. Each mutant was tested for proteolytic stability and transcription activity in vivo , and for DNA binding activity in vitro . Mutants of TraR with single substitutions at positions W184, V187, K189, E193Q, V197 and D217 have wild-type levels of accumulation and DNA binding, but are defective in transcription of both types of promoters. These residues constitute a patch on the surface of the DNA-binding domain. We propose that this patch is an activating region that recruits RNA polymerase to TraR-dependent promoters through direct contact. As residues of this patch are critical for activation at both a class I and a class II promoter, we predict that these residues may contact the C-terminal domain of the RNA polymerase a a a a -subunit.

Summary Hydroxyproline (Hyp) in decaying organic matter is a rich source of carbon and nitrogen for microorganisms. A bacterial pathway for Hyp catabolism is known; however, genes and function relationships are not established. In the pathway, trans‐4‐hydroxy‐l‐proline (4‐l‐Hyp) is epimerized to cis‐4‐hydroxy‐d‐proline (4‐d‐Hyp), and then, in three enzymatic reactions, the d‐isomer is converted via Δ‐pyrroline‐4‐hydroxy‐2‐carboxylate (HPC) and α‐ketoglutarate semialdehyde (KGSA) to α‐ketoglutarate (KG). Here a transcriptional analysis of cells growing on 4‐l‐Hyp, and the regulation and functions of genes from a Hyp catabolism locus of the legume endosymbiont Sinorhizobium meliloti are reported. Fourteen hydroxyproline catabolism genes (hyp), in five transcripts hypR, hypD, hypH, hypST and hypMNPQO(RE)XYZ, were negatively regulated by hypR. hypRE was shown to encode 4‐hydroxyproline 2‐epimerase and a hypRE mutant grew with 4‐d‐Hyp but not 4‐l‐Hyp. hypO, hypD and hypH are predicted to encode 4‐d‐Hyp oxidase, HPC deaminase and α‐KGSA dehydrogenase respectively. The functions for hypS, hypT, hypX, hypY and hypZ remain to be determined. The data suggest 4‐Hyp is converted to the tricarboxylic acid cycle intermediate α‐ketoglutarate via the pathway established biochemically for Pseudomonas. This report describes the first molecular characterization of a Hyp catabolism locus.

Hydroxyproline-rich proteins in plants offer a source of carbon and nitrogen to soil-dwelling microorganisms in the form of root exudates and decaying organic matter. This report describes an ABC-type transport system dedicated to the uptake of hydroxyproline in the legume endosymbiont Sinorhizobium meliloti. We have designated genes involved in hydroxyproline metabolism as hyp genes and show that an S. meliloti strain lacking putative transport genes (DeltahypMNPQ) is unable to grow with or transport trans-4-hydroxy-l-proline when this compound is available as a sole source of carbon. Expression of hypM is upregulated in the presence of trans-4-hydroxy-l-proline and cis-4-hydroxy-d-proline, as modulated by a repressor (HypR) of the GntR/FadR subfamily. Although alfalfa root nodules contain hydroxyproline-rich proteins, we demonstrate that the transport system is not highly expressed in nodules, suggesting that bacteroids are not exposed to high levels of free hydroxyproline in planta. In addition to hypMNPQ, we report that S. meliloti encodes a second independent mechanism that enables transport of trans-4-hydroxy-l-proline. This secondary transport mechanism is induced in proline-grown cells and likely entails a system involved in l-proline uptake. This study represents the first genetic description of a prokaryotic hydroxyproline transport system, and the ability to metabolize hydroxyproline may contribute significantly toward the ecological success of plant-associated bacteria such as the rhizobia.

No abstract