Agrobacterium tumefaciens fitness genes involved in the colonization of plant tumors and roots.

Agrobacterium tumefaciens colonizes the galls (plant tumors) it causes, and the roots of host and nonhost plants. Transposon-sequencing (Tn-Seq) was used to discover A.tumefaciens genes involved in reproductive success (fitness genes) on Solanum lycopersicum and Populus trichocarpa tumors and S.lycopersicum and Zea mays roots. The identified fitness genes represent 3-8% of A. tumefaciens genes and contribute to carbon and nitrogen metabolism, synthesis and repair of DNA, RNA and proteins and envelope-associated functions. Competition assays between 12 knockout mutants and wild-type confirmed the involvement of 10 genes (trpB, hisH, metH, cobN, ntrB, trxA, nrdJ, kamA, exoQ, wbbL) in A.tumefaciens fitness under both tumor and root conditions. The remaining two genes (fecA, noxA) were important in tumors only. None of these mutants was nonpathogenic, but four (hisH, trpB, exoQ, ntrB) exhibited impaired virulence. Finally, we used this knowledge to search for chemical and biocontrol treatments that target some of the identified fitness pathways and report reduced tumorigenesis and impaired establishment of A.tumefaciens on tomato roots using tannic acid or Pseudomonas protegens, which affect iron assimilation. This work revealed A.tumefaciens pathways that contribute to its competitive survival in plants and highlights a strategy to identify plant protection approaches against this pathogen.

Insight in the quorum sensing-driven lifestyle of the non-pathogenic Agrobacterium tumefaciens 6N2 and the interactions with the yeast Meyerozyma guilliermondii.

Agrobacterium tumefaciens is considered a prominent phytopathogen, though most isolates are nonpathogenic. Agrobacteria can inhabit plant tissues interacting with other microorganisms. Yeasts are likewise part of these communities. We analyzed the quorum sensing (QS) systems of A. tumefaciens strain 6N2, and its relevance for the interaction with the yeast Meyerozyma guilliermondii, both sugarcane endophytes. We show that strain 6N2 is nonpathogenic, produces OHC8-HSL, OHC10-HSL, OC12-HSL and OHC12-HSL as QS signals, and possesses a complex QS architecture, with one truncated, two complete systems, and three additional QS-signal receptors. A proteomic approach showed differences in QS-regulated proteins between pure (64 proteins) and dual (33 proteins) cultures. Seven proteins were consistently regulated by quorum sensing in pure and dual cultures. M. guilliermondii proteins influenced by QS activity were also evaluated. Several up- and down- regulated proteins differed depending on the bacterial QS. These results show the QS regulation in the bacteria-yeast interactions.

Import pathways of the mannityl-opines into the bacterial pathogen Agrobacterium tumefaciens: structural, affinity and in vivo approaches.

Agrobacterium tumefaciens pathogens use specific compounds denoted opines as nutrients in their plant tumor niche. These opines are produced by the host plant cells genetically modified by agrobacteria. They are imported into bacteria via solute-binding proteins (SBPs) in association with ATP-binding cassette transporters. The mannityl-opine family encompasses mannopine, mannopinic acid, agropine and agropinic acid. Structural and affinity data on mannopinic acid bound to SBPs are currently lacking while those of the three others mannityl opines are available. We investigated the molecular basis of two pathways for mannopinic acid uptake. MoaA was proposed as the specific SBP for mannopinic acid import in mannityl opines-assimilating agrobacteria, which was validated here using genetic studies and affinity measurements. We structurally characterized the mannopinic acid-binding mode of MoaA in two crystal forms at 2.05 and 1.57 Šresolution. We demonstrated that the non-specific SBP MotA, so far characterized as mannopine and Amadori compound importer, was also able to transport mannopinic acid. The structure of MotA bound to mannopinic acid at 2.2 Šresolution defines a different mannopinic acid-binding signature, similar to that of mannopine. Combining in vitro and in vivo approaches, this work allowed us to complete the characterization of the mannityl-opines assimilation pathways, highlighting the important role of two dual imports of agropinic and mannopinic acids. Our data shed new light on how the mannityl-opines contribute to the establishment of the ecological niche of agrobacteria from the early to the late stages of tumor development.

Structural basis for two efficient modes of agropinic acid opine import into the bacterial pathogen Agrobacterium tumefaciens.

Agrobacterium tumefaciens pathogens genetically modify their host plants to drive the synthesis of opines in plant tumors. The mannityl-opine family encompasses mannopine, mannopinic acid, agropine and agropinic acid. These opines serve as nutrients and are imported into bacteria via periplasmic-binding proteins (PBPs) in association with ABC transporters. Structural and affinity data on agropine and agropinic acid opines bound to PBPs are currently lacking. Here, we investigated the molecular basis of AgtB and AgaA, proposed as the specific PBP for agropine and agropinic acid import, respectively. Using genetic approaches and affinity measurements, we identified AgtB and its transporter as responsible for agropine uptake in agropine-assimilating agrobacteria. Nonetheless, we showed that AgtB binds agropinic acid with a higher affinity than agropine, and we structurally characterized the agropinic acid-binding mode through three crystal structures at 1.4, 1.74 and 1.9 Šresolution. In the crystallization time course, obtaining a crystal structure of AgtB with agropine was unsuccessful due to the spontaneous lactamization of agropine into agropinic acid. AgaA binds agropinic acid only with a similar affinity in nanomolar range as AgtB. The structure of AgaA bound to agropinic acid at 1.65 Šresolution defines a different agropinic acid-binding signature. Our work highlights the structural and functional characteristics of two efficient agropinic acid assimilation pathways, of which one is also involved in agropine assimilation.

From Intracellular Bacteria to Differentiated Bacteroids: Transcriptome and Metabolome Analysis in Aeschynomene Nodules Using the Bradyrhizobium sp. Strain ORS285 bclA Mutant.

Soil bacteria called rhizobia trigger the formation of root nodules on legume plants. The rhizobia infect these symbiotic organs and adopt an intracellular lifestyle within the nodule cells, where they differentiate into nitrogen-fixing bacteroids. Several legume lineages force their symbionts into an extreme cellular differentiation, comprising cell enlargement and genome endoreduplication. The antimicrobial peptide transporter BclA is a major determinant of this process in Bradyrhizobium sp. strain ORS285, a symbiont of Aeschynomene spp. In the absence of BclA, the bacteria proceed until the intracellular infection of nodule cells, but they cannot differentiate into enlarged polyploid and functional bacteroids. Thus, the bclA nodule bacteria constitute an intermediate stage between the free-living soil bacteria and the nitrogen-fixing bacteroids. Metabolomics on whole nodules of Aeschynomene afraspera and Aeschynomene indica infected with the wild type or the bclA mutant revealed 47 metabolites that differentially accumulated concomitantly with bacteroid differentiation. Bacterial transcriptome analysis of these nodules demonstrated that the intracellular settling of the rhizobia in the symbiotic nodule cells is accompanied by a first transcriptome switch involving several hundred upregulated and downregulated genes and a second switch accompanying the bacteroid differentiation, involving fewer genes but ones that are expressed to extremely elevated levels. The transcriptomes further suggested a dynamic role for oxygen and redox regulation of gene expression during nodule formation and a nonsymbiotic function of BclA. Together, our data uncover the metabolic and gene expression changes that accompany the transition from intracellular bacteria into differentiated nitrogen-fixing bacteroids.IMPORTANCE Legume-rhizobium symbiosis is a major ecological process, fueling the biogeochemical nitrogen cycle with reduced nitrogen. It also represents a promising strategy to reduce the use of chemical nitrogen fertilizers in agriculture, thereby improving its sustainability. This interaction leads to the intracellular accommodation of rhizobia within plant cells of symbiotic organs, where they differentiate into nitrogen-fixing bacteroids. In specific legume clades, this differentiation process requires the bacterial transporter BclA to counteract antimicrobial peptides produced by the host. Transcriptome analysis of Bradyrhizobium wild-type and bclA mutant bacteria in culture and in symbiosis with Aeschynomene host plants dissected the bacterial transcriptional response in distinct phases and highlighted functions of the transporter in the free-living stage of the bacterial life cycle.

Saline Environments as a Source of Potential Quorum Sensing Disruptors to Control Bacterial Infections: A Review.

Saline environments, such as marine and hypersaline habitats, are widely distributed around the world. They include sea waters, saline lakes, solar salterns, or hypersaline soils. The bacteria that live in these habitats produce and develop unique bioactive molecules and physiological pathways to cope with the stress conditions generated by these environments. They have been described to produce compounds with properties that differ from those found in non-saline habitats. In the last decades, the ability to disrupt quorum-sensing (QS) intercellular communication systems has been identified in many marine organisms, including bacteria. The two main mechanisms of QS interference, i.e., quorum sensing inhibition (QSI) and quorum quenching (QQ), appear to be a more frequent phenomenon in marine aquatic environments than in soils. However, data concerning bacteria from hypersaline habitats is scarce. Salt-tolerant QSI compounds and QQ enzymes may be of interest to interfere with QS-regulated bacterial functions, including virulence, in sectors such as aquaculture or agriculture where salinity is a serious environmental issue. This review provides a global overview of the main works related to QS interruption in saline environments as well as the derived biotechnological applications.

Transcriptomic dissection of Bradyrhizobium sp. strain ORS285 in symbiosis with Aeschynomene spp. inducing different bacteroid morphotypes with contrasted symbiotic efficiency.

To circumvent the paucity of nitrogen sources in the soil legume plants establish a symbiotic interaction with nitrogen-fixing soil bacteria called rhizobia. During symbiosis, the plants form root organs called nodules, where bacteria are housed intracellularly and become active nitrogen fixers known as bacteroids. Depending on their host plant, bacteroids can adopt different morphotypes, being either unmodified (U), elongated (E) or spherical (S). E- and S-type bacteroids undergo a terminal differentiation leading to irreversible morphological changes and DNA endoreduplication. Previous studies suggest that differentiated bacteroids display an increased symbiotic efficiency (E > U and S > U). In this study, we used a combination of Aeschynomene species inducing E- or S-type bacteroids in symbiosis with Bradyrhizobium sp. ORS285 to show that S-type bacteroids present a better symbiotic efficiency than E-type bacteroids. We performed a transcriptomic analysis on E- and S-type bacteroids formed by Aeschynomene afraspera and Aeschynomene indica nodules and identified the bacterial functions activated in bacteroids and specific to each bacteroid type. Extending the expression analysis in E- and S-type bacteroids in other Aeschynomene species by qRT-PCR on selected genes from the transcriptome analysis narrowed down the set of bacteroid morphotype-specific genes. Functional analysis of a selected subset of 31 bacteroid-induced or morphotype-specific genes revealed no symbiotic phenotypes in the mutants. This highlights the robustness of the symbiotic program but could also indicate that the bacterial response to the plant environment is partially anticipatory or even maladaptive. Our analysis confirms the correlation between differentiation and efficiency of the bacteroids and provides a framework for the identification of bacterial functions that affect the efficiency of bacteroids.© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

Lifestyle of the biotroph Agrobacterium tumefaciens in the ecological niche constructed on its host plant.

Agrobacterium tumefaciens constructs an ecological niche in its host plant by transferring the T-DNA from its Ti plasmid into the host genome and by diverting the host metabolism. We combined transcriptomics and genetics for understanding the A. tumefaciens lifestyle when it colonizes Arabidopsis thaliana tumors. Transcriptomics highlighted: a transition from a motile to sessile behavior that mobilizes some master regulators (Hfq, CtrA, DivK and PleD); a remodeling of some cell surface components (O-antigen, succinoglucan, curdlan, att genes, putative fasciclin) and functions associated with plant defense (Ef-Tu and flagellin pathogen-associated molecular pattern-response and glycerol-3-phosphate and nitric oxide signaling); and an exploitation of a wide variety of host resources, including opines, amino acids, sugars, organic acids, phosphate, phosphorylated compounds, and iron. In addition, construction of transgenic A. thaliana lines expressing a lactonase enzyme showed that Ti plasmid transfer could escape host-mediated quorum-quenching. Finally, construction of knock-out mutants in A. tumefaciens showed that expression of some At plasmid genes seemed more costly than the selective advantage they would have conferred in tumor colonization. We provide the first overview of A. tumefaciens lifestyle in a plant tumor and reveal novel signaling and trophic interplays for investigating host-pathogen interactions.

Structural Basis for High Specificity of Amadori Compound and Mannopine Opine Binding in Bacterial Pathogens.

Agrobacterium tumefaciens pathogens genetically modify their host plants to drive the synthesis of opines in plant tumors. Opines are either sugar phosphodiesters or the products of condensed amino acids with ketoacids or sugars. They are Agrobacterium nutrients and imported into the bacterial cell via periplasmic-binding proteins (PBPs) and ABC-transporters. Mannopine, an opine from the mannityl-opine family, is synthesized from an intermediate named deoxy-fructosyl-glutamine (DFG), which is also an opine and abundant Amadori compound (a name used for any derivative of aminodeoxysugars) present in decaying plant materials. The PBP MotA is responsible for mannopine import in mannopine-assimilating agrobacteria. In the nopaline-opine type agrobacteria strain, SocA protein was proposed as a putative mannopine binding PBP, and AttC protein was annotated as a mannopine binding-like PBP. Structural data on mannityl-opine-PBP complexes is currently lacking. By combining affinity data with analysis of seven x-ray structures at high resolution, we investigated the molecular basis of MotA, SocA, and AttC interactions with mannopine and its DFG precursor. Our work demonstrates that AttC is not a mannopine-binding protein and reveals a specific binding pocket for DFG in SocA with an affinity in nanomolar range. Hence, mannopine would not be imported into nopaline-type agrobacteria strains. In contrast, MotA binds both mannopine and DFG. We thus defined one mannopine and two DFG binding signatures. Unlike mannopine-PBPs, selective DFG-PBPs are present in a wide diversity of bacteria, including Actinobacteria, α-,ß-, and γ-proteobacteria, revealing a common role of this Amadori compound in pathogenic, symbiotic, and opportunistic bacteria.

Transcriptome analysis revealed that a quorum sensing system regulates the transfer of the pAt megaplasmid in Agrobacterium tumefaciens.

BACKGROUND: Agrobacterium tumefaciens strain P4 is atypical, as the strain is not pathogenic and produces a for this species unusual quorum sensing signal, identified as N-(3-hydroxy-octanoyl)-homoserine lactone (3OH,C8-HSL). RESULTS: By sequence analysis and cloning, a functional luxI-like gene, named cinI, has been identified on the At plasmid of A. tumefaciens strain P4. Insertion mutagenesis in the cinI gene and transcriptome analyses permitted the identification of 32 cinI-regulated genes in this strain, most of them encoding proteins responsible for the conjugative transfer of pAtP4. Among these genes were the avhB genes that encode a type 4 secretion system (T4SS) involved in the formation of the conjugation apparatus, the tra genes that encode the DNA transfer and replication (Dtr) machinery and cinI and two luxR orthologs. These last two genes, cinR and cinX, exhibit an unusual organization, with the cinI gene surrounded by the two luxR orthologs. Conjugation experiments confirmed that the conjugative transfer of pAtP4 is regulated by 3OH,C8-HSL. Root colonization experiments indicated that the quorum sensing regulation of the conjugation of the pAtP4 does not confer a gain or a loss of fitness to the bacterial host in the tomato plant rhizosphere. CONCLUSION: This work is the first identification of the occurrence of a quorum sensing regulation of the pAt conjugation phenomenon in Agrobacterium.

Agrobacterium uses a unique ligand-binding mode for trapping opines and acquiring a competitive advantage in the niche construction on plant host.

By modifying the nuclear genome of its host, the plant pathogen Agrobacterium tumefaciens induces the development of plant tumours in which it proliferates. The transformed plant tissues accumulate uncommon low molecular weight compounds called opines that are growth substrates for A. tumefaciens. In the pathogen-induced niche (the plant tumour), a selective advantage conferred by opine assimilation has been hypothesized, but not experimentally demonstrated. Here, using genetics and structural biology, we deciphered how the pathogen is able to bind opines and use them to efficiently compete in the plant tumour. We report high resolution X-ray structures of the periplasmic binding protein (PBP) NocT unliganded and liganded with the opine nopaline (a condensation product of arginine and α-ketoglurate) and its lactam derivative pyronopaline. NocT exhibited an affinity for pyronopaline (K(D) of 0.6 µM) greater than that for nopaline (KD of 3.7 µM). Although the binding-mode of the arginine part of nopaline/pyronopaline in NocT resembled that of arginine in other PBPs, affinity measurement by two different techniques showed that NocT did not bind arginine. In contrast, NocT presented specific residues such as M117 to stabilize the bound opines. NocT relatives that exhibit the nopaline/pyronopaline-binding mode were only found in genomes of the genus Agrobacterium. Transcriptomics and reverse genetics revealed that A. tumefaciens uses the same pathway for assimilating nopaline and pyronopaline. Fitness measurements showed that NocT is required for a competitive colonization of the plant tumour by A. tumefaciens. Moreover, even though the Ti-plasmid conjugal transfer was not regulated by nopaline, the competitive advantage gained by the nopaline-assimilating Ti-plasmid donors led to a preferential horizontal propagation of this Ti-plasmid amongst the agrobacteria colonizing the plant-tumour niche. This work provided structural and genetic evidences to support the niche construction paradigm in bacterial pathogens.

Concerted transfer of the virulence Ti plasmid and companion At plasmid in the Agrobacterium tumefaciens-induced plant tumour.

The plant pathogen Agrobacterium tumefaciens C58 harbours three independent type IV secretion (T4SS) machineries. T4SST-DNA promotes the transfer of the T-DNA to host plant cells, provoking tumour development and accumulation of opines such as nopaline and agrocinopines. T4SSpTi and T4SSpAt control the bacterial conjugation of the Ti and At plasmids respectively. Expression of T4SSpTi is controlled by the agrocinopine-responsive transcriptional repressor AccR. In this work, we compared the genome-wide transcriptional profile of the wild-type A. tumefaciens strain C58 with that of its accR KO-mutant to delineate the AccR regulon. In addition to the genes that encode agrocinopine catabolism and T4SSpTi , we found that AccR also regulated genes coding for nopaline catabolism and T4SSpAt . Further opine detection and conjugation assays confirmed the enhancement of nopaline consumption and At plasmid conjugation frequency in accR. Moreover, co-regulation of the T4SSpTi and T4SSpAt correlated with the co-transfer of the At and Ti plasmids both in vitro and in plant tumours. Finally, unlike T4SSpTi , T4SSpAt activation does not require quorum-sensing. Overall this study highlights the regulatory interplays between opines, At and Ti plasmids that contribute to a concerted dissemination of the two replicons in bacterial populations colonizing the plant tumour.

An increasing opine carbon bias in artificial exudation systems and genetically modified plant rhizospheres leads to an increasing reshaping of bacterial populations.

To investigate how exudation shapes root-associated bacterial populations, transgenic Arabidopsis thaliana plants that exuded the xenotopic compound octopine at low and high rates were grown in a nonsterile soil. Enumerations of both cultivable and octopine-degrading bacteria demonstrated that the ratios of octopine degraders increased along with octopine concentration. An artificial exudation system was also set up in which octopine was brought at four ratios. The density of octopine-degrading bacteria directly correlated with the input of octopine. Bacterial diversity was analysed by rrs amplicon pyrosequencing. Ensifer and Pseudomonas were significantly more frequently detected in soil amended with artificial exudates. However, the density of Pseudomonas increased as a response to carbon supplementation while that of Ensifer only correlated with octopine concentrations possibly in relation to two opposed colonization strategies of rhizosphere bacteria, that is, copiotrophy and oligotrophy.

Azospirillum genomes reveal transition of bacteria from aquatic to terrestrial environments.

Fossil records indicate that life appeared in marine environments ∼3.5 billion years ago (Gyr) and transitioned to terrestrial ecosystems nearly 2.5 Gyr. Sequence analysis suggests that "hydrobacteria" and "terrabacteria" might have diverged as early as 3 Gyr. Bacteria of the genus Azospirillum are associated with roots of terrestrial plants; however, virtually all their close relatives are aquatic. We obtained genome sequences of two Azospirillum species and analyzed their gene origins. While most Azospirillum house-keeping genes have orthologs in its close aquatic relatives, this lineage has obtained nearly half of its genome from terrestrial organisms. The majority of genes encoding functions critical for association with plants are among horizontally transferred genes. Our results show that transition of some aquatic bacteria to terrestrial habitats occurred much later than the suggested initial divergence of hydro- and terrabacterial clades. The birth of the genus Azospirillum approximately coincided with the emergence of vascular plants on land.

N,N'-alkylated Imidazolium-derivatives act as quorum-sensing inhibitors targeting the Pectobacterium atrosepticum-induced symptoms on potato tubers.

Bacteria belonging to the Pectobacterium genus are the causative agents of the blackleg and soft-rot diseases that affect potato plants and tubers worldwide. In Pectobacterium, the expression of the virulence genes is controlled by quorum-sensing (QS) and N-acylhomoserine lactones (AHLs). In this work, we screened a chemical library of QS-inhibitors (QSIs) and AHL-analogs to find novel QSIs targeting the virulence of Pectobacterium. Four N,N'-bisalkylated imidazolium salts were identified as QSIs; they were active at the µM range. In potato tuber assays, two of them were able to decrease the severity of the symptoms provoked by P. atrosepticum. This work extends the range of the QSIs acting on the Pectobacterium-induced soft-rot disease.

Heavy-metal resistance of a France vineyard soil bacterium, Pseudomonas mendocina strain S5.2, revealed by whole-genome sequencing.

Here we present the draft genome of Pseudomonas mendocina strain S5.2, possessing tolerance to a high concentration of copper. In addition to being copper resistant, the genome of P. mendocina strain S5.2 contains a number of heavy-metal-resistant genes known to confer resistance to multiple heavy-metal ions.

Catabolic pathway of gamma-caprolactone in the biocontrol agent Rhodococcus erythropolis.

Gamma-caprolactone (GCL) is well-known as a food flavor and has been recently described as a biostimulant molecule promoting the growth of bacteria with biocontrol activity against soft-rot pathogens. Among these biocontrol agents, Rhodococcus erythropolis, characterized by a remarkable metabolic versatility, assimilates various γ-butyrolactone molecules with a branched-aliphatic chain, such as GCL. The assimilative pathway of GCL in R. erythropolis was investigated by two-dimensional gel electrophoresis coupled to matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) analysis. This analysis suggests the involvement of the lactonase QsdA in ring-opening, a feature confirmed by heterologous expression in Escherichia coli. According to proteome analysis, the open-chain form of GCL was degraded by ß- and ω-oxidation coupled to the Krebs cycle and ß-ketoadipate pathway. Ubiquity of qsdA gene among environmental R. erythropolis isolates was verified by PCR. In addition to a previous N-acyl homoserine lactone catabolic function, QsdA may therefore be involved in an intermediate degradative step of cyclic recalcitrant molecules or in synthesis of flavoring lactones.

Identification of traits shared by rhizosphere-competent strains of fluorescent pseudomonads.

Rhizosphere competence of fluorescent pseudomonads is a prerequisite for the expression of their beneficial effects on plant growth and health. To date, knowledge on bacterial traits involved in rhizosphere competence is fragmented and derived mostly from studies with model strains. Here, a population approach was taken by investigating a representative collection of 23 Pseudomonas species and strains from different origins for their ability to colonize the rhizosphere of tomato plants grown in natural soil. Rhizosphere competence of these strains was related to phenotypic traits including: (1) their carbon and energetic metabolism represented by the ability to use a wide range of organic compounds, as electron donors, and iron and nitrogen oxides, as electron acceptors, and (2) their ability to produce antibiotic compounds and N-acylhomoserine lactones (N-AHSL). All these data including origin of the strains (soil/rhizosphere), taxonomic identification, phenotypic cluster based on catabolic profiles, nitrogen dissimilating ability, siderovars, susceptibility to iron starvation, antibiotic and N-AHSL production, and rhizosphere competence were submitted to multiple correspondence analyses. Colonization assays revealed a significant diversity in rhizosphere competence with survival rates ranging from approximately 0.1 % to 61 %. Multiple correspondence analyses indicated that rhizosphere competence was associated with siderophore-mediated iron acquisition, substrate utilization, and denitrification. However, the catabolic profile of one rhizosphere-competent strain differed from the others and its competence was associated with its ability to produce antibiotics phenazines and N-AHSL. Taken together, these data suggest that competitive strains have developed two types of strategies to survive in the rhizosphere.

Development and validation of a UHPLC-ESI-QTOF mass spectrometry method to analyze opines, plant biomarkers of crown gall or hairy root diseases.

Opines are low-molecular-weight metabolites specifically biosynthesized by agrobacteria-transformed plant cells when plants are struck by crown gall and hairy root diseases, which cause uncontrolled tissue overgrowth. Transferred DNA is sustainably incorporated into the genomes of the transformed plant cells, so that opines constitute a persistent biomarker of plant infection by pathogenic agrobacteria and can be targeted for crown gall/hairy root disease diagnosis. We developed a general, rapid, specific and sensitive analytical method for overall opine detection using ultra-high-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-MS-QTOF), with easy preparation of samples. Based on MS, MS/MS and chromatography data, the detection selectivity of a wide range of standard opines was validated in pure solution and in different plant extracts. The method was successfully used to detect different structural types of opines, including opines for which standard compounds are unavailable, in tumors or hairy roots induced by pathogenic strains. As the method can detect a wide range of opines in a single run, it represents a powerful tool for plant gall analysis and crown gall/hairy root disease diagnosis. Using an appropriate dilution of plant extract and a matrix-based calibration curve, the quantification ability of the method was validated for three opines belonging to different families (nopaline, octopine, mannopine), which were accurately quantified in plant tissue extracts.

Bradyrhizobium diazoefficiens USDA110 Nodulation of Aeschynomene afraspera Is Associated with Atypical Terminal Bacteroid Differentiation and Suboptimal Symbiotic Efficiency.

Legume plants can form root organs called nodules where they house intracellular symbiotic rhizobium bacteria. Within nodule cells, rhizobia differentiate into bacteroids, which fix nitrogen for the benefit of the plant. Depending on the combination of host plants and rhizobial strains, the output of rhizobium-legume interactions varies from nonfixing associations to symbioses that are highly beneficial for the plant. Bradyrhizobium diazoefficiens USDA110 was isolated as a soybean symbiont, but it can also establish a functional symbiotic interaction with Aeschynomene afraspera In contrast to soybean, A. afraspera triggers terminal bacteroid differentiation, a process involving bacterial cell elongation, polyploidy, and increased membrane permeability, leading to a loss of bacterial viability while plants increase their symbiotic benefit. A combination of plant metabolomics, bacterial proteomics, and transcriptomics along with cytological analyses were used to study the physiology of USDA110 bacteroids in these two host plants. We show that USDA110 establishes a poorly efficient symbiosis with A. afraspera despite the full activation of the bacterial symbiotic program. We found molecular signatures of high levels of stress in A. afraspera bacteroids, whereas those of terminal bacteroid differentiation were only partially activated. Finally, we show that in A. afraspera, USDA110 bacteroids undergo atypical terminal differentiation hallmarked by the disconnection of the canonical features of this process. This study pinpoints how a rhizobium strain can adapt its physiology to a new host and cope with terminal differentiation when it did not coevolve with such a host.IMPORTANCE Legume-rhizobium symbiosis is a major ecological process in the nitrogen cycle, responsible for the main input of fixed nitrogen into the biosphere. The efficiency of this symbiosis relies on the coevolution of the partners. Some, but not all, legume plants optimize their return on investment in the symbiosis by imposing on their microsymbionts a terminal differentiation program that increases their symbiotic efficiency but imposes a high level of stress and drastically reduces their viability. We combined multi-omics with physiological analyses to show that the symbiotic couple formed by Bradyrhizobium diazoefficiens USDA110 and Aeschynomene afraspera, in which the host and symbiont did not evolve together, is functional but displays a low symbiotic efficiency associated with a disconnection of terminal bacteroid differentiation features.