Emanuela Torelli

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.

The influence of local factors on the prediction of fumonisin contamination in maize

BACKGROUND Contamination by mycotoxins is a major concern to the maize industry in north-east Italy where maize grain is often spoiled by Fusarium spp. In this work, fumonisins, deoxynivalenol and zearalenone were determined and an artificial neural network (ANN) model suitable for predicting mycotoxin contamination of maize at harvest time was developed. RESULTS The occurrence of deoxynivalenol and zearalenone was very limited, while fumonisins concentration ranged from 163 and to 3663 µg kg(-1) in 2007, and from 333 to 11473 µg kg(-1) in 2008. Statistical data analysis of factors affecting fumonisins concentration revealed that irrigation, chemical treatment against the European corn borer and harvest date significantly affected the level of contamination (P < 0.05), although the relevance of the factors was different in 2007 and 2008. The neural network approach showed a significant correlation between ascertained values and predictions based on agronomic data. CONCLUSION This is the first time that an artificial neural network has been used to predict fumonisin accumulation in maize: the prediction has been shown to have the potential for the development of a new approach for the rapid cataloging of grain lots.

Air analysis in the assessment of fumonisin contamination risk in maize

BACKGROUND In maize-growing areas where fumonisin contamination is endemic, there is an urgent need for novel methods to assess the quality of grain lots before their delivery to common drying and storage collection centres. Aerobiological samples of fungal spores released during harvest were analysed to establish a relationship between fumonisin contamination and the abundance of pathogen propagules collected in the combine harvester using a cyclone and membrane filters. Filter-captured propagules were analysed by direct plating, immunoenzymatic assay of specific Fusarium extracellular polysaccharides and real time polymerase chain reaction of the extracted DNA using fum1, a gene involved in the biosynthesis of fumonisin, as a target. RESULTS The results showed that time of harvest and environmental conditions strongly influenced the efficiency and performance of the collection system. The data obtained were informative in comparing individual samples collected under similar conditions. The immunoenzymatic assay provided the most reliable data, which improved the ability of a neural network to predict the fumonisin content of lots, when added to agronomic, environmental and phytosanitary data. CONCLUSION This is the first attempt to evaluate the Fusarium propagules dispersed during harvesting as a predictive means to assess maize quality. A method based on cyclone/filter capture and immunological detection has been shown to be feasible and to have the potential for the development of a continuous monitoring system, but the prediction capabilities in the present implementation were limited.

Draft genome of a Xanthomonas perforans strain associated with pith necrosis

Xanthomonas perforans causes bacterial spot of tomato and pepper. A genome draft of an unusual isolate (strain 4P1S2), differing in that it was associated with stem pith necrosis, was assembled from Illumina MiSeq sequencing data using the draft of X. perforans strain 91-118 as a reference. The resulting draft (accession number JRWW00000000) largely overlapped with the reference draft. In addition, the reads not mapping on the reference assembly were selected and used for a further assembly, that revealed a large putative plasmid. The analysis of the predicted proteins showed only few gene features that could be potentially implicated in the switch of a phytopathological behavior.

Draft Genome Sequence of a Hypersensitive Reaction-Inducing Pantoea agglomerans Strain Isolated from Olive Knots Caused by Pseudomonas savastanoi pv. savastanoi

ABSTRACT Pantoea agglomerans strains inducing a hypersensitive reaction in tobacco leaves are frequently isolated inside olive knots caused by Pseudomonas savastanoi pv. savastanoi. Here, we report the draft genome sequence of the Italian P. agglomerans strain, which is able to increase olive knot disease severity when coinoculated with P. savastanoi pv. savastanoi.

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.

Isothermal folding of a light-up bio-orthogonal RNA origami nanoribbon

RNA presents intringuing roles in many cellular processes and its versatility underpins many different applications in synthetic biology. Nonetheless, RNA origami as a method for nanofabrication is not yet fully explored and the majority of RNA nanostructures are based on natural pre-folded RNA. Here we describe a biologically inert and uniquely addressable RNA origami scaffold that self-assembles into a nanoribbon by seven staple strands. An algorithm is applied to generate a synthetic De Bruijn scaffold sequence that is characterized by the lack of biologically active sites and repetitions larger than a predetermined design parameter. This RNA scaffold and the complementary staples fold in a physiologically compatible isothermal condition. In order to monitor the folding, we designed a new split Broccoli aptamer system. The aptamer is divided into two nonfunctional sequences each of which is integrated into the 5′ or 3′ end of two staple strands complementary to the RNA scaffold. Using fluorescence measurements and in-gel imaging, we demonstrate that once RNA origami assembly occurs, the split aptamer sequences are brought into close proximity forming the aptamer and turning on the fluorescence. This light-up ‘bio-orthogonal’ RNA origami provides a prototype that can have potential for in vivo origami applications.

Genome Plasticity and Dynamic Evolution of Phytopathogenic Pseudomonads and Related Bacteria

Pseudomonads represent widely distributed plant pathogenic bacteria, which cause diseases in most cultivated mono and dicotyledonous crops worldwide. Currently, 25 phytopathogenic Pseudomonas spp. have been identified and described; four of these species contain distinct pathovars. The general features of Pseudomonas syringae and Pseudomonas savastanoi include a genome size of approximately 6 Mb, with a 57–59 % G + C content, 5,200–5,700 protein-encoding genes, and 85.6–88.7 % coding sequences. The core genome of the P. syringae species complex contains 3,397 genes, whereas its “pan” genome, currently based on the genomic assessment of 19 different pathovars, contains 12,749 genes. Many strains of P. syringae pathovars contain one or more plasmids with an extensive amount of shared DNA sequences. There are striking examples of how the acquisition or loss of an entire plasmid or genomic (pathogenicity) island harboured from a particular plasmid determines a differential response of the host plant to bacterial attack. Cases of evolution through the gain or loss of a whole plasmid or its part are illustrated. Examples of dynamic genetic rearrangements involving genes for elicitors of the plant immunity system, the type III secretion system, and their effector proteins are illustrated along with a case of pathogen convergent evolution to the same host plant and the evolutionary dynamics found in Ralstonia solanacearum. Finally, cell-to-cell communication and two-component signal transduction are discussed in the global network of regulated communication.