Rapid and Efficient Methods to Isolate, Type Strains and Determine Species of Agrobacterium spp. in Pure Culture and Complex Environments

International audienc

Impact of Paraburkholderia phytofirmans PsJN on Grapevine Phenolic Metabolism

Phenolic compounds are implied in plant-microorganisms interaction and may be induced in response to plant growth-promoting rhizobacteria (PGPRs). Among PGPR, the beneficial bacterium Paraburkholderia phytofirmans PsJN was previously described to stimulate the growth of plants and to induce a better adaptation to both abiotic and biotic stresses. This study aimed to investigate the impact of PsJN on grapevine secondary metabolism. For this purpose, gene expression (qRT-PCR) and profiling of plant secondary metabolites (UHPLC-UV/DAD-MS QTOF) from both grapevine root and leaves were compared between non-bacterized and PsJN-bacterized grapevine plantlets. Our results showed that PsJN induced locally (roots) and systemically (leaves) an overexpression of PAL and STS and specifically in leaves the overexpression of all the genes implied in phenylpropanoid and flavonoid pathways. Moreover, the metabolomic approach revealed that relative amounts of 32 and 17 compounds in roots and leaves, respectively, were significantly modified by PsJN. Once identified to be accumulated in response to PsJN by the metabolomic approach, antifungal properties of purified molecules were validated in vitro for their antifungal effect on Botrytis cinerea spore germination. Taking together, our findings on the impact of PsJN on phenolic metabolism allowed us to identify a supplementary biocontrol mechanism developed by this PGPR to induce plant resistance against pathogens

Genomic Species Are Ecological Species as Revealed by Comparative Genomics in Agrobacterium tumefaciens

The definition of bacterial species is based on genomic similarities, giving rise to the operational concept of genomic species, but the reasons of the occurrence of differentiated genomic species remain largely unknown. We used the Agrobacterium tumefaciens species complex and particularly the genomic species presently called genomovar G8, which includes the sequenced strain C58, to test the hypothesis of genomic species having specific ecological adaptations possibly involved in the speciation process. We analyzed the gene repertoire specific to G8 to identify potential adaptive genes. By hybridizing 25 strains of A. tumefaciens on DNA microarrays spanning the C58 genome, we highlighted the presence and absence of genes homologous to C58 in the taxon. We found 196 genes specific to genomovar G8 that were mostly clustered into seven genomic islands on the C58 genome—one on the circular chromosome and six on the linear chromosome—suggesting higher plasticity and a major adaptive role of the latter. Clusters encoded putative functional units, four of which had been verified experimentally. The combination of G8-specific functions defines a hypothetical species primary niche for G8 related to commensal interaction with a host plant. This supports that the G8 ancestor was able to exploit a new ecological niche, maybe initiating ecological isolation and thus speciation. Searching genomic data for synapomorphic traits is a powerful way to describe bacterial species. This procedure allowed us to find such phenotypic traits specific to genomovar G8 and thus propose a Latin binomial, Agrobacterium fabrum, for this bona fide genomic species

Modulation des propriétés symbiotiques de Frankia (mécanismes de régulation génétique et métabolisme de l'azote)

Lors de cette étude, nous avons sélectionné différents gènes candidats impliqués dans le métabolisme de l'azote et la régulation de l'expression des gènes, activités qui semblent importantes pour l'établissement et le fonctionnement de la symbiose entre Frankia et les plantes actinorhiziennes. Ces gènes ont été étudiés sous deux angles, celui de leur histoire évolutive et de l'effet de l'hôte végétal sur leur diversification, et celui de leur rôle dans l'établissement et le fonctionnement des symbioses. Nous avons étudié les gènes glnII et RpoD, gènes impliqués respectivement dans l'assimilation de l'azote fixé et dans la régulation de la transcription. Ces deux gènes ont été utilisés pour évaluer les relations phylogénétiques entre les bactéries Frankia. Nous avons ainsi observé que les souches infectieuses de Frankia étaient divisées en deux groupes selon les spécificités de colonisation des plantes. Nous avons par ailleurs montré qu'au moins deux facteurs sigma primaires étaient retrouvés chez Frankia. L'un d'eux n'a pas été observé pour des souches isolées d'Elaeagnus ou d'Hippophae, ce qui pourrait refléter une spécialisation liée au partenaire végétal. Dans la continuité de cette approche qui consiste à étudier le déterminisme génétique impliqué dans les relations entre Frankia et les plantes actinorhiziennes, nous nous sommes intéressés aux systèmes de régulation à deux composants, et à l'activité de fixation d'azote. Nous avons montré que Frankia possédait des séquences homologues du gène régulateur gacA, et nous avons mis en évidence la présence d'au moins quatre gènes présentant des séquences homologues de senseurs de la famille EnvZ. Concernant l'activité de fixation d'azote, nous avons caractérisé une partie de l'îlot génétique nif de Frankia ACN14a, et montré que cette bactérie semble exprimer constitutivement les gènes nif, même en présence d'azote et sans différenciation de vésicules. Enfin, pour permettre la mise au point d'outils pouvant faciliter les études de génétique de Frankia, nous avons caractérisé un plasmide cryptique de Frankia ACN14a.LYON1-BU.Sciences (692662101) / SudocSudocFranceF

Back to basics : Rethinking the diversity and pathogeny of the causal agent of Bacterial Leaf Spot of Lettuce

International audienc

The plant defense signal galactinol is specifically used as a nutrient by the bacterial pathogen Agrobacterium fabrum

The bacterial plant pathogen Agrobacterium fabrum uses periplasmic-binding proteins (PBPs) along with ABC transporters to import a wide variety of plant molecules as nutrients. Nonetheless, how A. fabrum acquires plant metabolites is incompletely understood. Using genetic approaches and affinity measurements, we identified here the PBP MelB and its transporter as being responsible for the uptake of the raffinose family of oligosaccharides (RFO), which are the most widespread d-galactose-containing oligosaccharides in higher plants. We also found that the RFO precursor galactinol, recently described as a plant defense molecule, is imported into Agrobacterium via MelB with nanomolar range affinity. Structural analyses and binding mode comparisons of the X-ray structures of MelB in complex with raffinose, stachyose, galactinol, galactose, and melibiose (a raffinose degradation product) revealed how MelB recognizes the nonreducing end galactose common to all these ligands and that MelB has a strong preference for a two-unit sugar ligand. Of note, MelB conferred a competitive advantage to A. fabrum in colonizing the rhizosphere of tomato plants. Our integrative work highlights the structural and functional characteristics of melibiose and galactinol assimilation by A. fabrum, leading to a competitive advantage for these bacteria in the rhizosphere. We propose that the PBP MelB, which is highly conserved among both symbionts and pathogens from Rhizobiace family, is a major trait in these bacteria required for early steps of plant colonization