A highly conserved ligand-binding site for AccA transporters of antibiotic and quorum-sensing regulator in Agrobacterium leads to a different specificity.

Plants genetically modified by the pathogenic Agrobacterium strain C58 synthesize agrocinopines A and B, whereas those modified by the pathogenic strain Bo542 produce agrocinopines C and D. The four agrocinopines (A, B, C and D) serve as nutrients by agrobacteria and signaling molecule for the dissemination of virulence genes. They share the uncommon pyranose-2-phosphate motif, represented by the l-arabinopyranose moiety in agrocinopines A/B and the d-glucopyranose moiety in agrocinopines C/D, also found in the antibiotic agrocin 84. They are imported into agrobacterial cytoplasm via the Acc transport system, including the solute-binding protein AccA coupled to an ABC transporter. We have previously shown that unexpectedly, AccA from strain C58 (AccAC58) recognizes the pyranose-2-phosphate motif present in all four agrocinopines and agrocin 84, meaning that strain C58 is able to import agrocinopines C/D, originating from the competitor strain Bo542. Here, using agrocinopine derivatives and combining crystallography, affinity and stability measurements, modeling, molecular dynamics, in vitro and vivo assays, we show that AccABo542 and AccAC58 behave differently despite 75% sequence identity and a nearly identical ligand binding site. Indeed, strain Bo542 imports only compounds containing the d-glucopyranose-2-phosphate moiety, and with a lower affinity compared with strain C58. This difference in import efficiency makes C58 more competitive than Bo542 in culture media. We can now explain why Agrobacterium/Allorhizobium vitis strain S4 is insensitive to agrocin 84, although its genome contains a conserved Acc transport system. Overall, our work highlights AccA proteins as a case study, for which stability and dynamics drive specificity.

Construction of a Transposon Mutant Library in the Pathogen Agrobacterium tumefaciens C58 and Identification of Genes Involved in Gall Niche Exploitation and Colonization.

Agrobacterium tumefaciens is a plant pathogen that causes crown gall disease on a wide range of host species by transferring and integrating a part of its own DNA (T-DNA) into the plant genome. The genes responsible of the above-mentioned processes are well characterized. However, a large number of the mechanisms involved in exploitation and colonization of the galls (also named plant tumors) remain unknown. Due to recent development of "transposon-sequencing" (Tn-Seq) techniques, a high-throughput screening and identification of the different genes involved in such mechanisms is now possible. In this chapter, we describe the detailed methodology used to construct a transposon library in A. tumefaciens and to conduct a Tn-Seq approach to discover genes involved in plant tumor exploitation and colonization.

The Pseudomonas syringae pv. tomato DC3000 PSPTO_0820 multidrug transporter is involved in resistance to plant antimicrobials and bacterial survival during tomato plant infection.

Multidrug resistance efflux pumps protect bacterial cells against a wide spectrum of antimicrobial compounds. PSPTO_0820 is a predicted multidrug transporter from the phytopathogenic bacterium Pseudomonas syringae pv. tomato DC3000. Orthologs of this protein are conserved within many Pseudomonas species that interact with plants. To study the potential role of PSPTO_0820 in plant-bacteria interaction, a mutant in this gene was isolated and characterized. In addition, with the aim to find the outer membrane channel for this efflux system, a mutant in PSPTO_4977, a TolC-like gene, was also analyzed. Both mutants were more susceptible to trans-cinnamic and chlorogenic acids and to the flavonoid (+)-catechin, when added to the culture medium. The expression level of both genes increased in the presence of (+)-catechin and, in the case of PSPTO_0820, also in response to trans-cinnamic acid. PSPTO_0820 and PSPTO_4977 mutants were unable to colonize tomato at high population levels. This work evidences the involvement of these two proteins in the resistance to plant antimicrobials, supporting also the importance of chlorogenic acid, trans-cinnamic acid, and (+)-catechin in the tomato plant defense response against P. syringae pv. tomato DC3000 infection.

Integrative and deconvolution omics approaches to uncover the Agrobacterium tumefaciens lifestyle in plant tumors.

Agrobacterium tumefaciens is a plant pathogen which provokes galls on roots and stems (crown-gall disease) and colonizes them. Two approaches combining omics were used to decipher the lifestyle of A. tumefaciens in plant tumors: an integrative approach when omics were used on A. tumefaciens cells collected from plant tumors, a deconvolution approach when omics were used on A. tumefaciens cells exploiting a single tumor metabolite in pure culture assay. This addendum highlights some recent results on the biotroph lifestyle of A. tumefaciens in plant tumors.

The biotroph Agrobacterium tumefaciens thrives in tumors by exploiting a wide spectrum of plant host metabolites.

Agrobacterium tumefaciens is a niche-constructing biotroph that exploits host plant metabolites. We combined metabolomics, transposon-sequencing (Tn-seq), transcriptomics, and reverse genetics to characterize A. tumefaciens pathways involved in the exploitation of resources from the Solanum lycopersicum host plant. Metabolomics of healthy stems and plant tumors revealed the common (e.g. sucrose, glutamate) and enriched (e.g. opines, γ-aminobutyric acid (GABA), γ-hydroxybutyric acid (GHB), pyruvate) metabolites that A. tumefaciens could use as nutrients. Tn-seq and transcriptomics pinpointed the genes that are crucial and/or upregulated when the pathogen grew on either sucrose (pgi, kdgA, pycA, cisY) or GHB (blcAB, pckA, eno, gpsA) as a carbon source. While sucrose assimilation involved the Entner-Doudoroff and tricarboxylic acid (TCA) pathways, GHB degradation required the blc genes, TCA cycle, and gluconeogenesis. The tumor-enriched metabolite pyruvate is at the node connecting these pathways. Using reverse genetics, we showed that the blc, pckA, and pycA loci were important for aggressiveness (tumor weight), proliferation (bacterial charge), and/or fitness (competition between the constructed mutants and wild-type) of A. tumefaciens in plant tumors. This work highlighted how a biotroph mobilizes its central metabolism for exploiting a wide diversity of resources in a plant host. It further shows the complementarity of functional genome-wide scans by transcriptomics and Tn-seq to decipher the lifestyle of a plant pathogen.

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.

The plant GABA signaling downregulates horizontal transfer of the Agrobacterium tumefaciens virulence plasmid.

In the tumor-inducing (Ti) Agrobacterium tumefaciens, quorum sensing activates the horizontal transfer of the virulent Ti plasmid. In pure culture, this process can be impaired by the A. tumefaciens BlcC lactonase, whose expression is induced by gamma-aminobutyrate (GABA). It was therefore hypothesized that host GABA content might modulate quorum sensing and virulence gene dissemination during A. tumefaciens infection. We examined GABA metabolism and transport in Arabidopsis thaliana tumors combining transcriptomic, metabolomic and histological approaches. In addition, using genetically modified plants and bacteria, we evaluated the impact of plant host GABA content on Ti plasmid dissemination. The results showed that GABA and free proline, which acts as an antagonist of GABA uptake in A. tumefaciens, accumulated in wild-type tumors relative to uninfected plant tissues. Moreover, comparisons of tumors induced on Col-0 and her1 plants showed that the increase in the plant GABA : proline ratio was associated with both the upregulated expression of the blcC gene and the decreased dissemination of Ti plasmid in tumor-colonizing A. tumefaciens populations. This work demonstrates experimentally that the variation in the GABA content in plant tumors can interfere with the dissemination of A. tumefaciens Ti plasmids, and therefore highlights plant GABA content as an important trait in the struggle against pathogenic bacteria.