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Agrobacterium pathogens of octopine-and nopaline-types force host plants to produce either octopine or nopaline compounds, which they use as nutrients. Two Agrobacterium ABC-transporters and their cognate periplasmic binding proteins (PBPs) OccJ and NocT import octopine and nopaline/octopine, respectively. Here, we show that both octopine transport and degradation confer a selective advantage to octopine-type A. tumefaciens when it colonizes plants. We report the X-ray structures of the unliganded PBP OccJ and its complex with octopine as well as a structural comparison with NocT and the related PBP LAO from Salmonella enterica, which binds amino acids (lysine, arginine and ornithine). We investigated the specificity of OccJ, NocT and LAO using several ligands such as amino acids, octopine, nopaline and octopine analogues. OccJ displays a high selectivity and nanomolar range affinity for octopine. Altogether, the structural and affinity data allowed to define an octopine binding signature in PBPs and to construct a OccJ mutant impaired in octopine binding, a selective octopine-binding NocT and a non-selective octopine-binding LAO by changing one single residue in these PBPs. We proposed the PBP OccJ as a major trait in the ecological specialization of octopine-type Agrobacterium pathogens when they colonize and exploit the plant host.

Opines are produced in plant cells transformed with the A. tumefaciens T-DNA. These opines can be used as nutrients by the phytopathogen and some of them act as signaling molecules controlling Ti plasmid dissemination through quorum-sensing. The present study aims at an enlarged understanding of the roles of opines during A. tumefaciens/plant host interactions. First, based on transcriptomic investigations, the agrocinopine-controled AccR regulon from A. tumefaciens C58 was thoroughly characterized. This one includes functions associated with (i) agrocinopines assimilation, (ii) nopaline assimilation, (iii) quorum-sensing and Ti plasmid conjugation, (iv) At plasmid conjugation. Moreover our analysis showed that co-regulation of Ti and At plasmid conjugations correlated with the co-transfer of the two replicons. In a second step using functional genetic and structural biology we quantified the selective advantage conferred to colonizing A. tumefaciens populations by the assimilation of the opines nopaline and octopine. The molecular basis which underlies this selective advantage, especially regarding the sensing and import of the two opines within the bacterial cytoplasm, were also described. Finally combining metabolomics and reverse genetic with in planta conjugation assays we demonstrated the opposite effects of GABA and opine/quorum-sensing signaling on the dissemination of Ti plasmids. In conclusion our results reveal the interlink between opine, quorum-sensing and GABA signaling during A. tumefaciens/plant host interactions. They noticeably highlight the impact of this interlink on host colonization and the dissemination of Ti and At plasmid genes which are critical for the virulence and the adaption of the bacteria to the tumor lifestyle., Les opines sont des molécules produites dans les cellules végétales transformées par l’ADN-T d’A. tumefaciens. Ces opines peuvent être utilisées comme nutriments par le phytopathogène et certaines d’entre elles agissent comme signaux moléculaires contrôlant la dissémination du plasmide de virulence Ti via la signalisation quorum-sensing. Le présent travail vise à une compréhension élargie du rôle des opines au cours des interactions A. tumefaciens-plantes hôtes. En se basant d’abord sur des analyses transcriptomiques, le régulon AccR d’A. tumefaciens C58, contrôlé par les opines agrocinopines, a été défini : celui-ci inclut des fonctions associées (i) à l’assimilation des agrocinopines, (ii) à l’assimilation de la nopaline, (iii) à la signalisation quorum-sensing et la conjugaison du plasmide Ti, (iv) à la conjugaison du plasmide At. La corrélation entre la co-régulation des conjugaisons des plasmides Ti et At et le co-transfert des deux réplicons a en outre été mise en évidence. Dans un second temps, associant des approches de génétique fonctionnelle à des travaux collaboratifs en biologie structurale, l’avantage sélectif conféré par les opines nopaline et octopine à A. tumefaciens au sein des tumeurs végétales a été quantifié. Les bases moléculaires sous-jacentes à cet avantage sélectif, notamment celles associées à la perception et l’importation des deux opines dans le cytoplasme bactérien, ont été décrites. Enfin, en combinant des approches de métabolomique et de génétique inverse avec des tests de conjugaison in planta, les effets opposés de la signalisation GABA d’une part et des signalisations opine et quorum-sensing d’autre part sur la dissémination du plasmide Ti ont été démontrés. En conclusion, nos résultats révèlent l’intrication des signalisations opine, quorum-sensing et GABA au cours de l’interaction A. tumefaciens-plantes hôtes. Ils soulignent en particulier les impacts de cette intrication sur la colonisation de l’hôte ainsi que sur la dissémination des gènes de virulence et d’adaptation à l’environnement tumeur portés par les plasmides Ti et At.

Laboratoire d'accueil : CNRS UPR 2355 Institut des Sciences du végétal (ISV) équipe Rhizosphère Gif sur Yvette; Les opines sont des molécules produites dans les cellules végétales transformées par l’ADN-T d’A. tumefaciens. Ces opines peuvent être utilisées comme nutriments par le phytopathogène et certaines d’entre elles agissent comme signaux moléculaires contrôlant la dissémination du plasmide de virulence Ti via la signalisation quorum-sensing. Le présent travail vise à une compréhension élargie du rôle des opines au cours des interactions A. tumefaciens-plantes hôtes. En se basant d’abord sur des analyses transcriptomiques, le régulon AccR d’A. tumefaciens C58, contrôlé par les opines agrocinopines, a été défini : celui-ci inclut des fonctions associées (i) à l’assimilation des agrocinopines, (ii) à l’assimilation de la nopaline, (iii) à la signalisation quorum-sensing et la conjugaison du plasmide Ti, (iv) à la conjugaison du plasmide At. La corrélation entre la co-régulation des conjugaisons des plasmides Ti et At et le co-transfert des deux réplicons a en outre été mise en évidence. Dans un second temps, associant des approches de génétique fonctionnelle à des travaux collaboratifs en biologie structurale, l’avantage sélectif conféré par les opines nopaline et octopine à A. tumefaciens au sein des tumeurs végétales a été quantifié. Les bases moléculaires sous-jacentes à cet avantage sélectif, notamment celles associées à la perception et l’importation des deux opines dans le cytoplasme bactérien, ont été décrites. Enfin, en combinant des approches de métabolomique et de génétique inverse avec des tests de conjugaison in planta, les effets opposés de la signalisation GABA d’une part et des signalisations opine et quorum-sensing d’autre part sur la dissémination du plasmide Ti ont été démontrés. En conclusion, nos résultats révèlent l’intrication des signalisations opine, quorum-sensing et GABA au cours de l’interaction A. tumefaciens-plantes hôtes. Ils soulignent en particulier les impacts de cette intrication sur la colonisation de l’hôte ainsi que sur la dissémination des gènes de virulence et d’adaptation à l’environnement tumeur portés par les plasmides Ti et At.

International audience; During the past ten years, emerging data revealed the role of E2F factors in various plant physiology aspects. E2F involvement in cell cycle and plant development was particularly investigated and highlighted some plant specificities, espacially in postembryonic development. A major role of E2F is its transcriptional activity governing specific gene induction throughout the cell cycle and also strong induction of DNA repair genes in response to DNA damage, notably in the context of DNA double strand break (DSB) response. More challenging is the implication of E2F in nuclear foci with gamma-H2AX, a marker of DSB, probably independently of its transcriptional activity, in the DNA damage response.

Some bacteria produce and perceive quorum-sensing (QS) signals that coordinate several behaviours, including the costly processes that are exoenzyme production and plasmid transfer. In the case of plasmid transfer, the emergence of QS signal-altered invaders and their policing are poorly documented. In Agrobacterium tumefaciens, the virulence Ti-plasmid encodes both synthesis and sensing of QS-signals, which promote its transfer from a donor to a recipient cell. Here, we reported that QS-altered A. tumefaciens mutants arose during experimental evolution. All showed improved growth compared to their ancestor. Genome sequencing revealed that, though some had lost the Ti-plasmid, most were defective for QS-signal synthesis and Ti-plasmid conjugation (traR mutations) and one exhibited a QS-signal exploitation behaviour, using signal produced by other cells to enhance its own Ti-plasmid transfer. We explored mechanisms that can limit this QS-hijacking. We showed that the A. tumefaciens capacity to inactivate QS-signals by expressing QS-degrading enzyme could attenuate dissemination of the QS signal-negative Ti-plasmids. This work shows that enzymatic QSdisruption whether encoded by the QS-producing Ti-plasmid itself, by a companion plasmid in the same donor cells, or by one in the recipient cells, in all cases can serve as a mechanism for controlling QS exploitation by QS signal-negative mutants.

International audience; In Agrobacterium tumefaciens, horizontal transfer and vegetative replication of oncogenic Ti plasmids involve a cell-to-cell communication process called quorum-sensing (QS). The determinants of the QS-system belong to the LuxR/Luxl class. The Luxl-like protein Tral synthesizes N-acyl-homoserine lactone molecules which act as diffusible QS-signals. Beyond a threshold concentration, these molecules bind and activate the LuxR-like transcriptional regulator TraR, thereby initiating the QS-regulatory pathway. For the last 20 years, A. tumefaciens has stood as a prominent model in the understanding of the LuxR/Lux1 type of QS systems. A number of studies also unveiled features which are unique to A. tumefaciens QS, some of them being directly related to the phytopathogenic lifestyle of the bacteria. In this review, we will present the current knowledge of QS in A. tumefaciens at both the genetic and molecular levels. We will also describe how interactions with plant host modulate the QS pathway of A. tumefaciens, and discuss what could be the advantages for the agrobacteria to use such a tightly regulated QS-system to disseminate the Ti plasmids.

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 (KD 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., Author Summary An ecological niche is defined, in a given environment, by the availability of nutritive resources, which can be specifically assimilated by certain living organisms to promote their proliferation. The bacterial pathogen Agrobacterium tumefaciens is able to engineer an ecological niche in the infected host via the transformation of the plant genome and diversion of the plant metabolism towards production of the opine nutrients. In this work, we quantified the selective advantage conferred to a member of the phytopathogenic species A. tumefaciens which is able to assimilate the opine nopaline. This opine is a condensate of arginine and α-ketoglurate that is produced both under linear and cyclic forms in the plant tumour environment. We further determined at the molecular and atomistic levels how A. tumefaciens is able to sense the nopaline molecules, and which metabolic pathways are activated in response. Overall, this work deciphered some key molecular events in the niche construction of the pathogen A. tumefaciens that is unique among living organisms and used to develop bioengineering tools.