lpxC and yafS are the Most Suitable Internal Controls to Normalize Real Time RT-qPCR Expression in the Phytopathogenic Bacteria Dickeya dadantii

Background: Quantitative RT-PCR is the method of choice for studying, with both sensitivity and accuracy, the expression of genes. A reliable normalization of the data, using several reference genes, is critical for an accurate quantification of gene expression. Here, we propose a set of reference genes, of the phytopathogenic bacteria Dickeya dadantii and Pectobacterium atrosepticum, which are stable in a wide range of growth conditions. [br/] Results: We extracted, from a D. dadantii micro-array transcript profile dataset comprising thirty-two different growth conditions, an initial set of 49 expressed genes with very low variation in gene expression. Out of these, we retained 10 genes representing different functional categories, different levels of expression (low, medium, and high) and with no systematic variation in expression correlating with growth conditions. We measured the expression of these reference gene candidates using quantitative RT-PCR in 50 different experimental conditions, mimicking the environment encountered by the bacteria in their host and directly during the infection process in planta. The two most stable genes (ABF-0017965 (lpxC) and ABF-0020529 (yafS) were successfully used for normalization of RT-qPCR data. Finally, we demonstrated that the ortholog of lpxC and yafS in Pectobacterium atrosepticum also showed stable expression in diverse growth conditions. [br/] Conclusions: We have identified at least two genes, lpxC (ABF-0017965) and yafS (ABF-0020509), whose expressions are stable in a wide range of growth conditions and during infection. Thus, these genes are considered suitable for use as reference genes for the normalization of real-time RT-qPCR data of the two main pectinolytic phytopathogenic bacteria D. dadantii and P. atrosepticum and, probably, of other Enterobacteriaceae. Moreover, we defined general criteria to select good reference genes in bacteria

Étude des activités biologiques interférant avec la signalisation bactérienne reposant sur les N-acylhomosérine lactones dans l'environnement rhizosphérique

L expression de nombreuses fonctions bactériennes est régulée selon un mécanisme, appelé quorum sensing (QS), qui permet l'expression synchronisée de gènes cibles au sein d'une population lorsque que celle-ci a atteint un certain seuil de densité. Ce système repose sur la production et la perception d'une molécule signal qui diffuse dans l'environnement. Chez diverses Protéobactéries de la rhizosphère (Agrobacterium, Rhizobiacea, Erwinias, etc.), la production des signaux QS de type N-acylhomosérine lactones (AHL) est associée à la régulation de fonctions essentielles dans l'interaction avec la plante : transfert de plasmide pathogène ou symbiotique, colonisation, nodulation, production d'enzymes lytiques, d'cxopolysaccharides, etc. Les organismes avoisinant les bactéries productrices d'AHL ont développé différents mécanismes interférant avec ce système : production de composés mimétiques ou antagonistes du signal (plantes, champignons), dégradation du signal (bactéries, champignons, animaux). M. truncatula et A. thaliana sont par ailleurs capables de percevoir les AHL et d exprimer en retour des réponses spécifiques. Les travaux présentés ici relatent principalement la caractérisation d'une nouvelle enzyme dégradant les signaux AHL (AHLase), chez des plantes de la famille des légumineuses (Papilionideae). La purification et l'analyse par HPLC/MS-MS du support protéique de l'activité AHL lactonase chez Vicia sativa indiquent que celui-ci correspond à une protéine apparentée à un type particulier de lipoxygénases (LOX) présent uniquement chez les légumineuses. Certaines AHL, exercent par ailleurs un effet inhibiteur sur l activité LOX. Parallèlement à cette étude moléculaire, une étude fonctionnelle a été initiée. Dans ce cadre, l'impact potentiel de l'expression d'une activité AHLase par une plante, naturellement non-dégadatrice des AHL (tabac), sur les communautés bactériennes rhizosphériques cultivables a été analysé. Si elle peut exercer un impact ciblé sur une bactérie QS-dépendante, l expression de l'activité AHLase ne semble pas, dans ce contexte, perturber la structure globale de la population bactérienne rhizosphérique. Les légumineuses forment des nodosités symbiotiques avec les Rhizobiaceae, bactéries présentant à ce jour les systèmes QS les plus complexes. Ces études mettent donc l'accent sur une inconnue fondamentale: le rôle exact des AHL dans la signalisation entre bactéries, et entre bactéries et plante, au cours de la symbiose. Elles s inscrivent par ailleurs dans un champ de recherche novateur et porteur de nombreuses perspectives, notamment dans le domaine du bio-contrôle contre les pathogènes QS-dépendants.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Transcriptome analysis of the Dickeya dadantii PecS regulon during early stages of interaction with Arabidopsis thaliana : D. dadantii in planta PecS regulon

PecS is one of the major global regulators controlling the virulence of Dickeya dadantii, a broad-host-range phytopathogenic bacterium causing soft rot on several plant families. To define the PecS regulon during plant colonization, we analysed the global transcriptome profiles in wild-type and pecS mutant strains during the early colonization of the leaf surfaces and in leaf tissue just before the onset of symptoms, and found that the PecS regulon consists of more than 600 genes. About one-half of these genes are down-regulated in the pecS mutant; therefore, PecS has both positive and negative regulatory roles that may be direct or indirect. Indeed, PecS also controls the regulation of a few dozen regulatory genes, demonstrating that this global regulator is at or near the top of a major regulatory cascade governing adaptation to growth in planta. Notably, PecS acts mainly at the very beginning of infection, not only to prevent virulence gene induction, but also playing an active role in the adaptation of the bacterium to the epiphytic habitat. Comparison of the patterns of gene expression inside leaf tissues and during early colonization of leaf surfaces in the wild-type bacterium revealed 637 genes modulated between these two environments. More than 40% of these modulated genes are part of the PecS regulon, emphasizing the prominent role of PecS during plant colonization

Fungal invasion of the rhizosphere microbiome

The rhizosphere is the infection court where soil-borne pathogens establish a parasitic relationship with the plant. To infect root tissue, pathogens have to compete with members of the rhizosphere microbiome for available nutrients and microsites. In disease-suppressive soils, pathogens are strongly restricted in growth by the activities of specific rhizosphere microorganisms. Here, we sequenced metagenomic DNA and RNA of the rhizosphere microbiome of sugar beet seedlings grown in a soil suppressive to the fungal pathogen Rhizoctonia solani. rRNA-based analyses showed that Oxalobacteraceae, Burkholderiaceae, Sphingobacteriaceae and Sphingomonadaceae were significantly more abundant in the rhizosphere upon fungal invasion. Metatranscriptomics revealed that stress-related genes (ppGpp metabolism and oxidative stress) were upregulated in these bacterial families. We postulate that the invading pathogenic fungus induces, directly or via the plant, stress responses in the rhizobacterial community that lead to shifts in microbiome composition and to activation of antagonistic traits that restrict pathogen infection

A Rhodococcus qsdA-Encoded Enzyme Defines a Novel Class of Large-Spectrum Quorum-Quenching Lactonases▿ †

A gene involved in N-acyl homoserine lactone (N-AHSL) degradation was identified by screening a genomic library of Rhodococcus erythropolis strain W2. This gene, named qsdA (for quorum-sensing signal degradation), encodes an N-AHSL lactonase unrelated to the two previously characterized N-AHSL-degrading enzymes, i.e., the lactonase AiiA and the amidohydrolase AiiD. QsdA is related to phosphotriesterases and constitutes the reference of a novel class of N-AHSL degradation enzymes. It confers the ability to inactivate N-AHSLs with an acyl chain ranging from C6 to C14, with or without substitution at carbon 3. Screening of a collection of 15 Rhodococcus strains and strains closely related to this genus clearly highlighted the relationship between the ability to degrade N-AHSLs and the presence of the qsdA gene in Rhodococcus. Bacteria harboring the qsdA gene interfere very efficiently with quorum-sensing-regulated functions, demonstrating that qsdA is a valuable tool for developing quorum-quenching procedures

Are soils suppressive to fungal diseases the sources of biocontrol agents ?

International audienceSoils suppressive to soil-borne diseases are defined by a low disease incidence in spite of the presence of a virulent pathogen and a susceptible plant. In many cases, the inhibition of the disease development relies on the activity of the resident soil microbiome. Suppressiveness can be transmitted to conducive soil by mixing a small amount of suppressive soil into the conducive one. To identify microbial taxons linked to the suppressive phenotype of soils, culture independent-based methods have been employed to analyze and compare microbial diversities in two different soils suppressive (respectively conducive) to either Fusarium wilt of flax or Rhizoctonia diseases of sugar beet. Metagenomic DNA was extracted from the rhizosphere of plants grown in these soils. Fungal and bacterial taxonomic diversity was estimated from ITS and 16S genes by amplicon pyrosequencing. Structural shifts were revealed among rhizosphere fungal communities in suppressive soils in absence and in presence of the pathogen, as well as in conducive soils. Important differences noticed in taxonomic composition between suppressive and conducive soils in each system suggest that different rhizosphere fungal groups are linked with disease suppression in Fusarium wilt and Rhizoctonia diseases. Comparisons of suppressive and conducive soils for a given disease revealed fungal groups that may harbour potential biocontrol agents. Fungal groups most abundant in the mix of soil than in conducive soil were identified for Fusarium wilt and for Rhizoctonia diseases respectively. In both cases, some fungi showed an increase in the suppressive soil in the presence of the pathogen (vs non-inoculated suppressive soil). Similar results were obtained for the bacterial communities. These results suggest the role of suppressive soils as sources of specific more than generalist biocontrol agents

Identifying indicators of soil suppressiveness to fungal diseases

National audienceSoils suppressive to soil-borne diseases are defined by a low disease incidence in spite of the presence of a virulent pathogen and a susceptible plant. In many cases, the inhibition of the disease development relies on the activity of the resident soil microbiome. To identify taxonomic microbial indicators linked to the suppressiveness phenotype of soils, culture independent-based methods have been employed to analyse and compare microbial dynamics in two different soils suppressive to either Rhizoctonia solani damping-off disease of sugar beet or Fusarium wilt disease on flax. Fungal and bacterial taxonomic biodiversity were estimated from ITS and 16S genes by amplicon pyrosequencing. To that end, metagenomic DNA was extracted from the rhizosphere of plants grown in soils with different level of suppressiveness. We obtained 218650 reads in total (125602 for fungi and 93048 for bacteria). At this moment, the analyses of fungal communities are in progress. 114641 reads was kept after filtering by bioinformatic pipeline, distributed into 2303 clusters and 3379 singletons. Although, the bioinformatic and statistical analysis are not finished yet, we have already noticed a difference in the taxonomic diversity composition between suppressive and conducive soils which could explain the suppressive/conducive character of given soil. The next step is to achieve the bacterial communities in Fusarium wilt suppressive/conducive soils and to assess the microbial diversity of other soil-borne diseases suppressive soils. Once the analyses of sequencing data are finished and the taxonomic assignments done, the comparison of microbial diversity of all studied soils will be performed in order to find out the similarities or/and differences in these soils which will provide the suppressiveness indicator

Comparative Microbiome Analysis of a Fusarium Wilt Suppressive Soil and a Fusarium Wilt Conducive Soil From the Châteaurenard Region

Disease-suppressive soils are soils in which specific soil-borne plant pathogens cause only limited disease although the pathogen and susceptible host plants are both present. Suppressiveness is in most cases of microbial origin. We conducted a comparative metabarcoding analysis of the taxonomic diversity of fungal and bacterial communities from suppressive and non-suppressive (conducive) soils as regards Fusarium wilts sampled from the Châteaurenard region (France). Bioassays based on Fusarium wilt of flax confirmed that disease incidence was significantly lower in the suppressive soil than in the conducive soil. Furthermore, we succeeded in partly transferring Fusarium wilt-suppressiveness to the conducive soil by mixing 10% (w/w) of the suppressive soil into the conducive soil. Fungal diversity differed significantly between the suppressive and conducive soils. Among dominant fungal operational taxonomic units (OTUs) affiliated to known genera, 17 OTUs were detected exclusively in the suppressive soil. These OTUs were assigned to the Acremonium, Chaetomium, Cladosporium, Clonostachys, Fusarium, Ceratobasidium, Mortierella, Penicillium, Scytalidium, and Verticillium genera. Additionally, the relative abundance of specific members of the bacterial community was significantly higher in the suppressive and mixed soils than in the conducive soil. OTUs found more abundant in Fusarium wilt-suppressive soils were affiliated to the bacterial genera Adhaeribacter, Massilia, Microvirga, Rhizobium, Rhizobacter, Arthrobacter, Amycolatopsis, Rubrobacter, Paenibacillus, Stenotrophomonas, and Geobacter. Several of the fungal and bacterial genera detected exclusively or more abundantly in the Fusarium wilt-suppressive soil included genera known for their activity against F. oxysporum. Overall, this study supports the potential role of known fungal and bacterial genera in Fusarium wilt suppressive soils from Châteaurenard and pinpoints new bacterial and fungal genera for their putative role in Fusarium wilt suppressiveness

Adhesion of Gastric Cancer Cells to the Enteric Nervous System: Comparison between the Intestinal Type and Diffuse Type of Gastric Cancer

Background: Gastric cancer (GC) is the third leading cause of cancer-related deaths worldwide. The enteric nervous system (ENS) has been suggested to be involved in cancer development and spread. Objective: To analyze the GC cell adhesion to the ENS in a model of co-culture of gastric ENS with GC cells. Methods: Primary culture of gastric ENS (pcgENS), derived from a rat embryo stomach, was developed. The adhesion of GC cells to pcgENS was studied using a co-culture model. The role of N-Cadherin, a cell-adhesion protein, was evaluated. Results: Compared to intestinal-type GC cells, the diffuse-type GC cancer cells showed higher adhesion to pcgENS (55.9% ± 1.075 vs. 38.9% ± 0.6611, respectively, p < 0.001). The number of diffuse-type GC cells adherent to pcgENS was significantly lower in neuron-free pcgENS compared to neuron-containing pcgENS (p = 0.0261 and 0.0329 for AGS and MKN45, respectively). Confocal microscopy showed that GC cells adhere preferentially to the neurons of the pcgENS. N-Cadherin blockage resulted in significantly decreased adhesion of the GC cells to the pcgENS (p < 0.01). Conclusion: These results suggest a potential role of enteric neurons in the dissemination of GC cells, especially of the diffuse-type, partly through N-Cadherin