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Aims: Crown gall, a phytobacteriosis characterized by the formation of tumours on plant roots was observed in recently planted vineyards of the Meknes region (Morocco). The objective of this research was to analyse the diversity of pathogenic agrobacteria isolated from grapevine in Morocco., Methods and Results: Eighty-two isolates from 11 affected vineyards were characterized by recA sequencing and were found to belong to Agrobacterium tumefaciens genomospecies G1, G4 or G7, Rhizobium rhizogenes, and to Allorhizobium vitis. Only the All. vitis isolates appeared to be pathogenic on tomato and multilocus sequence analysis phylogenetic analyses revealed a weak genetic diversity, with the definition of only four genomic groups. Definition of the All. vitis genomic groups correlated with specific pathogenic traits: indeed, genomic groups differed with respect to the severity of hypersensitive response symptoms on tobacco leaves, the intensity of necrotic response on grapevine explants and opine profiles. Both vitopine and octopine were detected by UHPLC in tumours induced by isolates of three genomic groups, an opine signature scarcely ever reported., Conclusions: Allorhizobium vitis is the only causative agent of crown gall on grape in Morocco, pathogenic isolates can be separated into four genomic groups., Significance and Impact of the Study: This study of recently crown-gall-infested vineyards demonstrated that All. vitis is the only causative agent and revealed the presence of nonpathogenic Agrobacterium strain within tumours. Moreover, as the genetic diversity of the All. vitis isolates is relatively narrow, this study lays the basis for further analyses on the evolution of the disease, on the dissemination of the pTi and more globally on the fate of the different genomic groups in this newly colonized environment., (© 2019 The Society for Applied Microbiology.)

GABA acts as an intercellular signal in eukaryotes and as an interspecies signal in host-microbe interactions. Structural characteristics of selective eukaryotic GABA receptors and bacterial GABA sensors are unknown. Here, we identified the selective GABA-binding protein, called Atu4243, in the plant pathogen Agrobacterium tumefaciens. A constructed atu4243 mutant was affected in GABA transport and in expression of the GABA-regulated functions, including aggressiveness on two plant hosts and degradation of the quorum-sensing signal. The GABA-bound Atu4243 structure at 1.28 Å reveals that GABA adopts a conformation never observed so far and interacts with two key residues, Arg203 and Asp226 of which the role in GABA binding and GABA signalling in Agrobacterium has been validated using appropriate mutants. The conformational GABA-analogue trans-4-aminocrotonic acid ( TACA) antagonizes GABA activity, suggesting structural similarities between the binding sites of the bacterial sensor Atu4243 and mammalian GABAC receptors. Exploration of genomic databases reveals Atu4243 orthologues in several pathogenic and symbiotic proteobacteria, such as Rhizobium, Azospirillum, Burkholderia and Pseudomonas. Thus, this study establishes a structural basis for selective GABA sensors and offers opportunities for deciphering the role of the GABA-mediated communication in several host-pathogen interactions. [ABSTRACT FROM AUTHOR]

Agrobacterium has evolved sophisticated strategies to perceive and transduce plant-derived cues. Recent studies have found that numerous plant signals, including salicylic acid (SA), indole-3-acetic acid (IAA) and γ-amino butyric acid (GABA), profoundly affect Agrobacterium–plant interactions. Here we determine and compare the transcriptome profiles of Agrobacterium in response to these three plant signals. Collectively, the transcription of 103, 115 and 95 genes was significantly altered by SA, IAA and GABA respectively. Both distinct cellular responses and overlapping signalling pathways were elicited by these three plant signals. Interestingly, these three plant compounds function additively to shut off the Agrobacterium virulence programme and activate the quorum-quenching machinery. Moreover, the repression of the virulence programme by SA and IAA and the inactivation of quorum-sensing signals by SA and GABA are regulated through independent pathways. Our data indicate that these plant signals, while cross-talk in plant signalling networks, also act as cross-kingdom signals and play redundant roles in tailoring Agrobacterium regulatory pathways, resulting in intensive signalling cross-talk in Agrobacterium. Our results support the notion that Agrobacterium has evolved the ability to hijack plant signals for its own benefit. The complex signalling interplay between Agrobacterium and its plant hosts reflects an exquisite co-evolutionary balance. [ABSTRACT FROM AUTHOR]

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Abstract   Agrobacterium rhizogenes is the etiological agent for hairy-root disease (also known as root-mat disease). This bacterium induces the neoplastic growth of plant cells that differentiate to form “hairy roots.” Morphologically, A. rhizogenes-induced hairy roots are very similar in structure to wild-type roots with a few notable exceptions: Root hairs are longer, more numerous, and root systems are more branched and exhibit an agravitropic phenotype. Hairy roots are induced by the incorporation of a bacterial-derived segment of DNA transferred (T-DNA) into the chromosome of the plant cell. The expression of genes encoded within the T-DNA promotes the development and production of roots at the site of infection on most dicotyledonous plants. A key characteristic of hairy roots is their ability to grow quickly in the absence of exogenous plant growth regulators. As a result, hairy roots are widely used as a transgenic tool for the production of metabolites and for the study of gene function in plants. Researchers have utilized this tool to study root development and root–biotic interactions, to overexpress proteins and secondary metabolites, to detoxify environmental pollutants, and to increase drought tolerance. In this review, we provide an up-to-date overview of the current knowledge of how A. rhizogenes induces root formation, on the new uses for A. rhizogenes in tissue culture and composite plant production (wild-type shoots with transgenic roots), and the recent development of a disarmed version of A. rhizogenes for stable transgenic plant production. [ABSTRACT FROM AUTHOR]

Eastern Mediterranean sediments are characterized by the occurrence of distinct, organic-rich layers, called sapropels. These harbour elevated microbial numbers in comparison with adjacent carbon-lean intermediate layers. A recently obtained culture collection from these sediments was composed of 20% of strains closely related to Rhizobium radiobacter, formerly classified as Agrobacterium tumefaciens. To prove and quantify the in situ abundance of R. radiobacter, a highly specific quantitative polymerase chain reaction (PCR) protocol was developed. To convert quantification results into cell numbers, the copy number of rrn operons per genome was determined. Southern hybridization showed that our isolates contained four operons. Finally, quantitative PCR was applied to 45 sediment samples obtained across the eastern Mediterranean. Rhizobium radiobacter was present in 38 of 45 samples indicating an almost ubiquitous distribution. In total, 25–40 000 cells per gram of sediment were detected, corresponding to 0.001–5.1% of the bacterial cells. In general, the relative and absolute abundance of R. radiobacter increased with depth and was higher in sapropels than in intermediate layers. This indicates that R. radiobacter forms an active population in up to 200 000 years old sapropels. The present study shows for the first time that a cultivated subsurface bacterium is highly abundant in this environment. [ABSTRACT FROM AUTHOR]

The basic replication unit of many plasmids and second chromosomes in the alpha-proteobacteria consists of a repABC locus that encodes the trans- and cis-acting components required for both semiautonomous replication and replicon maintenance in a cell population. In terms of physical genetic organization and at the nucleotide sequence level, repABC loci are well conserved across various genera. As with all repABC-type replicons that have been genetically characterized, the 1.4 Mb pSymA and 1.7 Mb pSymB megaplasmids from the plant endosymbiont Sinorhizobium meliloti encode strong incompatibility ( inc) determinants. We have identified a novel inc sequence upstream of the repA2 gene in pSymA that is not present on pSymB and not reported in other repABC plasmids that have been characterized. This region, in concert with the repA and repB genes, stabilizes a test plasmid indicating that it constitutes a partitioning ( par) system for the megaplasmid. Purified RepB binds to this sequence and binding may be enhanced by RepA. We have isolated 19 point mutations that eliminate incompatibility, reduce RepB binding or the stabilization phenotype associated with this sequence and all of these map to a 16-nucleotide palindromic sequence centred 330 bp upstream of the repA2 gene. An additional five near-perfect repeats of this palindrome are located further upstream of the repA2 gene and we show that they share some conservation with known RepB binding sites in different locations on other repABC plasmids and to two sequences found on the tumour inducing plasmid of Agrobacterium tumefaciens. These additional palindromes also bind RepB but one of them does not display obvious incompatibility effects. A heterogenic distribution of par sequences demonstrates unexpected diversity in the structural genetic organization of repABC loci, despite their obvious levels of similarity. [ABSTRACT FROM AUTHOR]

Plasmids of Agrobacterium tumefaciens replicate using the products of the repABC operon, which are highly conserved among plasmids and some chromosomes of the alpha-Proteobacteria. The products of repA and repB direct plasmid partitioning, while the repC gene encodes a replication initiator protein. The transcription of the repABC operon of tumour inducing (Ti) plasmids is both negatively autoregulated by the RepA and RepB proteins, and positively regulated by TraR. In the present study, we have identified a fourth gene ( repD) in the repABC operon of an octopine-type Ti plasmid. repD is 78 codons in length, and maps between repA and repB genes. A repD–lacZ protein fusion demonstrated that repD is strongly expressed. Two identical binding sites for the RepB protein were found within the repD coding sequence, and these sites are required for plasmid stability and for maximal repression of repABC transcription. RepA protein enhances the binding of RepB at these binding sites, just as RepB increases the affinity of RepA for binding sites at the repABC P4 promoter. We propose that RepA and RepB form complexes that bind both sites, possibly causing a loop that is important for repression of the repABC operon. Binding at one or both sites may also be required for accurate plasmid partitioning. [ABSTRACT FROM AUTHOR]

Agrobacterium-mediated transient assays for gene function are increasingly being used as alternatives to genetic complementation and stable transformation. However, such assays are variable and not equally successful in different plant species. We analysed a range of genetic and physiological factors affecting transient expression following agroinfiltration, and developed a protocol for efficient and routine transient assays in several plant species. Lettuce exhibited high levels of transient expression and was at least as easy to work with asNicotiana benthamiana. Transient expression occurred in the majority of cells within the infiltrated tissue and approached 100% in some regions. High levels of transient expression were obtained in some ecotypes ofArabidopsis; however,Arabidopsisremains recalcitrant to routine, genotype-independent transient assays. Transient expression levels often exceeded those observed in stably transformed plants. The laboratoryAgrobacterium tumefaciensstrain C58C1 was the best strain for use in plant species that did not elicit a necrotic response toA. tumefaciens. A wildA. tumefaciensstrain, 1D1246, was identified that provided high levels of transient expression in solanaceous plants without background necrosis, enabling routine transient assays in these species. [ABSTRACT FROM AUTHOR]

The crdS gene of Agrobacterium sp. strain ATCC31749 encodes the curdlan synthase (CrdS) protein based on the homology of the derived CrdS protein sequence with those of β-glycosyl transferases with repetitive action patterns (Stasinopoulos et al. [1999] Glycobiology, 9, 31-41). Here we show that chemical (NTG) mutagenesis of crdS abolishes curdlan production and the induced mutations can be complemented by a cloned crdS amplicon, thus providing genetic confirmation that crdS is essential for curdlan production. When expressed in the native Agrobacterium or in Escherichia coli, the largely hydrophobic CrdS protein exhibited an Mr of ∼60 kDa (compared with the predicted mass of 73,121 Da) and was located in the inner membrane of both bacteria. By analyzing reciprocal fusions between crdS and the reporter genes, lacZ and phoA, and assessing the sensitivity of CrdS in spheroplasts to proteinase K, CrdS was shown to be an integral membrane protein with seven transmembrane helices and an Nout-Cin disposition. A central large and relatively hydrophilic cytoplasmic region carries the substrate-binding and catalytic D,D,D35QxxRW motif. The amino acid sequence of this domain of CrdS was threaded onto the 3D structure of the comparable domain of the SpsA protein, a member of the family GT-2 glycosyl transferases, and enabled the identification of corresponding amino acids involved in binding UDP in CrdS. Analysis of Agrobacterium membrane preparations using blue native-PAGE provided preliminary evidence that CrdS occurs in multimeric protein complexes of ∼420 kDa and ∼500 kDa. [ABSTRACT FROM AUTHOR]

Chemoreceptor (also called methyl-accepting chemotaxis protein, MCP) is the leading signal protein in the chemotaxis signaling pathway. MCP senses and binds chemoeffectors, specifically, and transmits the sensed signal to downstream proteins of the chemotaxis signaling system. The genome of Agrobacterium fabrum (previously, tumefaciens) C58 predicts that a total of 20 genes can encode MCP, but only the MCP-encoding gene atu0514 is located inside the che operon. Hence, the identification of the exact function of atu0514-encoding chemoreceptor (here, named as MCP514) will be very important for us to understand more deeply the chemotaxis signal transduction mechanism of A. fabrum. The deletion of atu0514 significantly decreased the chemotactic migration of A. fabrum in a swim plate. The test of atu0514-deletion mutant (Δ514) chemotaxis toward single chemicals showed that the deficiency of MCP514 significantly weakened the chemotactic response of A. fabrum to four various chemicals, sucrose, valine, citric acid and acetosyringone (AS), but did not completely abolish the chemotactic response. MCP514 was localized at cell poles although it lacks a transmembrane (TM) region and is predicted to be a cytoplasmic chemoreceptor. The replacement of residue Phe328 showed that the helical structure in the hairpin subdomain of MCP514 is a direct determinant for the cellular localization of MCP514. Single respective replacements of key residues indicated that residues Asn336 and Val353 play a key role in maintaining the chemotactic function of MCP514. [ABSTRACT FROM AUTHOR]

The pathogen Agrobacterium induces gall formation on a wide range of dicotyledonous plants. In this bacteria, most pathogenicity determinants are borne on the tumour inducing (Ti) plasmid. The conjugative transfer of this plasmid between agrobacteria is regulated by quorum sensing (QS). However, processes involved in the disturbance of QS also occur in this bacteria under the molecular form of a protein, TraM, inhibiting the sensing of the QS signals, and two lactonases BlcC (AttM) and AiiB that degrade the acylhomoserine lactone (AHL) QS signal. In the model Agrobacteriumfabrum strain C58, several data, once integrated, strongly suggest that the QS regulation may not be reacting only to cell concentration. Rather, these QS elements in association with the quorum quenching (QQ) activities may constitute an integrated and complex “go/no go system” that finely controls the biologically costly transfer of the Ti plasmid in response to multiple environmental cues. This decision mechanism permits the bacteria to sense whether it is in a gall or not, in a living or decaying tumor, in stressed plant tissues, etc. In this scheme, the role of the lactonases selected and maintained in the course of Ti plasmid and agrobacterial evolution appears to be pivotal. [ABSTRACT FROM AUTHOR]