Genome Sequence and Antifungal Activity of Two Niche-Sharing Pseudomonas protegens Related Strains Isolated from Hydroponics.

This work reports the comparison of the genome sequence and the ability to inhibit fungal growth of two Pseudomonas protegens related strains that were isolated from the same hydroponic culture of lamb's lettuce. The two strains were very similar in their core genome but one strain, Pf4, contained three gene clusters for the production of secondary metabolites, i.e., pyoluteorin (plt), pyrrolnitrin (prn), and rhizoxin (rzx), that were missing in the other strain, Pf11. The difference between the two strains was not due to simple insertion events, but to a relatively complex differentiation focused on the accessory genomes. In dual culture assays, both strains inhibited nearly all tested fungal strains, yet Pf4 exerted a significantly stronger fungal growth inhibition than Pf11. In addition to the differences in the secondary metabolite production associated genes abundance, the genome of Pf4 was more stable, smaller in size and with a lower number of transposons. The preservation of a dynamic equilibrium within natural populations of different strains comprised in the same species but differing in their secondary metabolite repertoire and in their genome stability may be functional to the adaptation to environmental changes.

Assembly of Phytoplasma Genome Drafts from Illumina Reads Using Phytoassembly.

Genome drafts for the phytoplasmas may be rapidly and efficiently assembled from NGS sequence data alone exploiting the proper bioinformatic tools and starting from properly collected samples. Here, we describe the use of the Phytoassembly pipeline ( https://github.com/cpolano/phytoassembly ), a fully automated tool that accepts as input row Illumina data from two samples (a phytoplasma infected sample and a healthy reference sample) to produce a phytoplasma genome draft, using the healthy plant host genome as a filter and profiting from the difference in reads coverage between the genome of the pathogen and that of the host. For phytoplasma infected samples containing >2% of pathogen DNA and an isogenic healthy reference sequence the resulting assemblies span the almost entire genomes.

An Effective Pipeline Based on Relative Coverage for the Genome Assembly of Phytoplasmas and Other Fastidious Prokaryotes.

BACKGROUND: For the plant pathogenic phytoplasmas, as well as for several fastidious prokaryotes, axenic cultivation is extremely difficult or not possible yet; therefore, even with second generation sequencing methods, obtaining the sequence of their genomes is challenging due to host sequence contamination. OBJECTIVE: With the Phytoassembly pipeline here presented, we aim to provide a method to obtain high quality genome drafts for the phytoplasmas and other uncultivable plant pathogens, by exploiting the coverage differential in the ILLUMINA sequences from the pathogen and the host, and using the sequencing of a healthy, isogenic plant as a filter. VALIDATION: The pipeline has been benchmarked using simulated and real ILLUMINA runs from phytoplasmas whose genome is known, and it was then used to obtain high quality drafts for three new phytoplasma genomes. CONCLUSION: For phytoplasma infected samples containing >2-4% of pathogen DNA and an isogenic reference healthy sample, the resulting assemblies can be next to complete. The Phytoassembly source code is available on GitHub at https://github.com/cpolano/phytoassembly.

Genomic Structural Variations Affecting Virulence During Clonal Expansion of Pseudomonas syringae pv. actinidiae Biovar 3 in Europe.

Pseudomonas syringae pv. actinidiae (Psa) biovar 3 caused pandemic bacterial canker of Actinidia chinensis and Actinidia deliciosa since 2008. In Europe, the disease spread rapidly in the kiwifruit cultivation areas from a single introduction. In this study, we investigated the genomic diversity of Psa biovar 3 strains during the primary clonal expansion in Europe using single molecule real-time (SMRT), Illumina and Sanger sequencing technologies. We recorded evidences of frequent mobilization and loss of transposon Tn6212, large chromosome inversions, and ectopic integration of IS sequences (remarkably ISPsy31, ISPsy36, and ISPsy37). While no phenotype change associated with Tn6212 mobilization could be detected, strains CRAFRU 12.29 and CRAFRU 12.50 did not elicit the hypersensitivity response (HR) on tobacco and eggplant leaves and were limited in their growth in kiwifruit leaves due to insertion of ISPsy31 and ISPsy36 in the hrpS and hrpR genes, respectively, interrupting the hrp cluster. Both strains had been isolated from symptomatic plants, suggesting coexistence of variant strains with reduced virulence together with virulent strains in mixed populations. The structural differences caused by rearrangements of self-genetic elements within European and New Zealand strains were comparable in number and type to those occurring among the European strains, in contrast with the significant difference in terms of nucleotide polymorphisms. We hypothesize a relaxation, during clonal expansion, of the selection limiting the accumulation of deleterious mutations associated with genome structural variation due to transposition of mobile elements. This consideration may be relevant when evaluating strategies to be adopted for epidemics management.

A DNA origami nanorobot controlled by nucleic acid hybridization.

A prototype for a DNA origami nanorobot is designed, produced, and tested. The cylindrical nanorobot (diameter of 14 nm and length of 48 nm) with a switchable flap, is able to respond to an external stimulus and reacts by a physical switch from a disarmed to an armed configuration able to deliver a cellular compatible message. In the tested design the robot weapon is a nucleic acid fully contained in the inner of the tube and linked to a single point of the internal face of the flap. Upon actuation the nanorobot moves the flap extracting the nucleic acid that assembles into a hemin/G-quadruplex horseradish peroxidase mimicking DNAzyme catalyzing a colorimetric reaction or chemiluminescence generation. The actuation switch is triggered by an external nucleic acid (target) that interacts with a complementary nucleic acid that is beard externally by the nanorobot (probe). Hybridization of probe and target produces a localized structural change that results in flap opening. The flap movement is studied on a two-dimensional prototype origami using Förster resonance energy transfer and is shown to be triggered by a variety of targets, including natural RNAs. The nanorobot has potential for in vivo biosensing and intelligent delivery of biological activators.