Erwinia amylovora Novel Plasmid pEI70: Complete Sequence, Biogeography, and Role in Aggressiveness in the Fire Blight Phytopathogen

Comparative genomics of several strains of Erwinia amylovora, a plant pathogenic bacterium causal agent of fire blight disease, revealed that its diversity is primarily attributable to the flexible genome comprised of plasmids. We recently identified and sequenced in full a novel 65.8 kb plasmid, called pEI70. Annotation revealed a lack of known virulence-related genes, but found evidence for a unique integrative conjugative element related to that of other plant and human pathogens. Comparative analyses using BLASTN showed that pEI70 is almost entirely included in plasmid pEB102 from E. billingiae, an epiphytic Erwinia of pome fruits, with sequence identities superior to 98%. A duplex PCR assay was developed to survey the prevalence of plasmid pEI70 and also that of pEA29, which had previously been described in several E. amylovora strains. Plasmid pEI70 was found widely dispersed across Europe with frequencies of 5–92%, but it was absent in E. amylovora analyzed populations from outside of Europe. Restriction analysis and hybridization demonstrated that this plasmid was identical in at least 13 strains. Curing E. amylovora strains of pEI70 reduced their aggressiveness on pear, and introducing pEI70 into low-aggressiveness strains lacking this plasmid increased symptoms development in this host. Discovery of this novel plasmid offers new insights into the biogeography, evolution and virulence determinants in E. amylovora

The Digital MIQE Guidelines Update: Minimum Information for Publication of Quantitative Digital PCR Experiments for 2020

Digital PCR (dPCR) has developed considerably since the publication of the Minimum Information for Publication of Digital PCR Experiments (dMIQE) guidelines in 2013, with advances in instrumentation, software, applications, and our understanding of its technological potential. Yet these developments also have associated challenges; data analysis steps, including threshold setting, can be difficult and preanalytical steps required to purify, concentrate, and modify nucleic acids can lead to measurement error. To assist independent corroboration of conclusions, comprehensive disclosure of all relevant experimental details is required. To support the community and reflect the growing use of dPCR, we present an update to dMIQE, dMIQE2020, including a simplified dMIQE table format to assist researchers in providing key experimental information and understanding of the associated experimental process. Adoption of dMIQE2020 by the scientific community will assist in standardizing experimental protocols, maximize efficient utilization of resources, and further enhance the impact of this powerful technology

Diversity within the novel Dickeya fangzhongdai sp., isolated from infected orchids, water and pears

International audienceMembers of the genus Dickeya are plant pathogens that mostly cause soft-rot diseases. Diversity within the genus has led to the recent description of three novel species (i.e. a mainly clonal D. solani of economic consequence in potatoes, D. aquatica from water samples and D. fangzhongdai isolated from necrotic pear trees in China). However, multilocus sequence analysis (MLSA) has highlighted the occurrence of seven isolates not related to the defined Dickeya species. These include a previously unclassified group of Dickeya strains isolated from monocotyledonous plants, Dickeya sp. S1, Dickeya sp. B16, and Dickeya sp. NCPPB 3274, as well as Dickeya sp. MK7, a strain isolated from water, and misassigned strains D. solani ND14, D. solani M005 and D. chrysanthemi M074. Here, it is reported that these isolates can be classified as D. fangzhongdai by using average nucleotide identity analysis and MLSA. However, variations in phenotypic characterization featuring colony morphology and carbon utilization assays revealed a diversity of bacterial isolates classified as D. fangzhongdai. Based on the results of the genomic analysis, the classification of Dickeya sp. S1, Dickeya sp. B16, Dickeya sp. MK7, Dickeya sp. NCPPB 3274 to D. fangzhongdai is proposed as well as the reclassification of D. solani ND14, D. solani M005 and D. chrysanthemi M074 to D. fangzhongdai

Falsification of Cyber-Physical Systems with Constrained Signal Spaces

International audienceFalsification has garnered much interest recently as a way to validate complex CPS designs with respect to a specification expressed via temporal logics. Using their quantitative semantics, the falsification problem can be formulated as robustness minimization problem. To make this infinite-dimensional problem tractable, a common approach is to restrict to classes of signals that can be defined using a finite number of parameters, such as piecewise-constant or piecewise-linear signals with fixed time intervals). A major drawback of this approach is that when the input signals must satisfy non-trivial temporal constraints, encoding these constraints into bounded domains for parameters can be difficult. In this work, to better capture temporal constraints on the input signal space, we use timed automata (TA) and make use of a transformation that allows sampling TA traces by sampling points in the unit box. We exploit this transformation to efficiently encode constrained CPS signals in the robustness minimization problem. This transformation also allows us to define an effective coverage measure of the constrained signal space so as to provide quantitative guarantees when no falsifying behaviour is found. In addition, this coverage is used to improve the black-box optimisation performance by detecting situations where the search is stuck near a local optimum. The approach is demonstrated on a ∆Σ modulator and a model of car automatic transmission subject to constraints describing usual driving patterns

Assessment of Digital PCR as a Primary Reference Measurement Procedure to Support Advances in Precision Medicine

BACKGROUND: Genetic testing of tumor tissue and circulating cell-free DNA for somatic variants guides patient treatment of many cancers. Such measurements will be fundamental in the future support of precision medicine. However, there are currently no primary reference measurement procedures available for nucleic acid quantification that would support translation of tests for circulating tumor DNA into routine use. METHODS: We assessed the accuracy of digital PCR (dPCR) for copy number quantification of a frequently occurring single-nucleotide variant in colorectal cancer (KRAS c.35GA, p.Gly12Asp, from hereon termed G12D) by evaluating potential sources of uncertainty that influence dPCR measurement. RESULTS: Concentration values for samples of KRAS G12D and wild-type plasmid templates varied by 1.2- fold when measured using 5 different assays with varying detection chemistry (hydrolysis, scorpion probes, and intercalating dyes) and 1.3-fold with 4 commercial dPCR platforms. Measurement trueness of a selected dPCR assay and platform was validated by comparison with an orthogonal method (inductively coupled plasma mass spectrometry). The candidate dPCR reference measurement procedure showed linear quantification over a wide range of copies per reaction and high repeatability and interlaboratory reproducibility (CV, 2%– 8% and 5%–10%, respectively). CONCLUSIONS: This work validates dPCR as an SItraceable reference measurement procedure based on enumeration and demonstrates how it can be applied for assignment of copy number concentration and fractional abundance values to DNA reference materials in an aqueous solution. High-accuracy measurements using dPCR will support the implementation and traceable standardization of molecular diagnostic procedures needed for advancements in precision medicine

VALITEST: Validation of diagnostic tests to support plant health

peer reviewedVALITEST is an EU-funded project built to improve the reliability of diagnostic tests performed in plant health laboratories across the European and Mediterranean region. The project is undertaken by a consortium of 16 partners composed of research institutions, private companies (such as diagnostic kit providers), national plant protection organizations and one intergovernmental organization (EPPO). Current harmonized procedures for the validation and organization of test performance studies will be improved based on the experience gained from the project and by including appropriate statistical approaches, by adapting the process for new promising technologies (e.g. high-throughput sequencing) and by providing new guidelines for the production of reference materials for validation studies. The project will provide a more complete and precise description of the performance of 82 diagnostic tests targeting 11 pests of interest for stakeholders of the region. It will also tackle the need for proficient users by developing a horizontal approach for the evaluation of laboratories’ proficiency and by organizing training activities on the concept of validation. The outcomes of the project will stimulate, optimize and strengthen the interactions between stakeholders in plant health for better diagnostics and lay the foundations for structuring the quality and the commercial offers for plant health diagnostics tools thanks to the creation of a dedicated association and a quality charter