Use of the GeneReader NGS System in a clinical pathology laboratory: a comparative study.

Despite its successful use in academic research, next-generation sequencing (NGS) still represents many challenges for routine clinical adoption due to its inherent complexity and specialised expertise typically required to set-up, test and operate a complete workflow.This study aims to evaluate QIAGEN's newly launched GeneReader NGS System solution in a pathology laboratory setting by assessing the system's ease of use, sequencing accuracy and data reproducibility. Our laboratory was able to implement the system and validate its performance using clinical samples in direct comparison to an approved Sanger sequencing platform and to an alternative in-house NGS technology. The QIAGEN workflow focuses on clinically actionable hotspots maximising testing efficiency. Combined with automated upstream sample processing and integrated downstream bioinformatics, it offers a realistic solution for pathology laboratories with limited prior experience in NGS technology.

KP4 to control Ustilago tritici in wheat: Enhanced greenhouse resistance to loose smut and changes in transcript abundance of pathogen related genes in infected KP4 plants.

Ustilago tritici causes loose smut, which is a seed-borne fungal disease of wheat, and responsible for yield losses up to 40%. Loose smut is a threat to seed production in developing countries where small scale farmers use their own harvest as seed material. The killer protein 4 (KP4) is a virally encoded toxin from Ustilago maydis and inhibits growth of susceptible races of fungi from the Ustilaginales. Enhanced resistance in KP4 wheat to stinking smut, which is caused by Tilletia caries, had been reported earlier. We show that KP4 in genetically engineered wheat increased resistance to loose smut up to 60% compared to the non-KP4 control under greenhouse conditions. This enhanced resistance is dose and race dependent. The overexpression of the transgene kp4 and its effect on fungal growth have indirect effects on the expression of endogenous pathogen defense genes.

Surveying of pollen-mediated crop-to-crop gene flow from a wheat field trial as a biosafety measure.

Outcrosses from genetically modified (GM) to conventional crops by pollen-mediated gene flow (PMGF) are a concern when growing GM crops close to non-GM fields. This also applies to the experimental releases of GM plants in field trials. Therefore, biosafety measures such as isolation distances and surveying of PMGF are required by the regulatory authorities in Switzerland. For two and three years, respectively, we monitored crop-to-crop PMGF from GM wheat field trials in two locations in Switzerland. The pollen donors were two GM spring wheat lines with enhanced fungal resistance and a herbicide tolerance as a selection marker. Seeds from the experimental plots were sampled to test the detection method for outcrosses. Two outcrosses were found adjacent to a transgenic plot within the experimental area. For the survey of PMGF, pollen receptor plots of the conventional wheat variety Frisal used for transformation were planted in the border crop and around the experimental field up to a distance of 200 m. Although the environmental conditions were favorable and the donor and receptor plots flowered at the same time, only three outcrosses were found in approximately 185,000 tested seedlings from seeds collected outside the experimental area. All three hybrids were found in the border crop surrounding the experimental area, but none outside the field. We conclude that a pollen barrier (border crop) and an additional isolation distance of 5 m is a sufficient measure to reduce PMGF from a GM wheat field trial to cleistogamous varieties in commercial fields below a level that can be detected.

Metabolomics and food processing: from semolina to pasta.

The objective of this study was to investigate the metabolite variations during industrial pasta processing (from semolina to dried pasta) for five different commercial products. Up to 76 metabolites were detected. Significant differences were observed between wholemeal and refined pasta samples, with the wholemeal pasta richer in many classes of compounds such as phytosterols, policosanols, unsaturated fatty acids, amino acids, carotenoids, minerals, and so on. Significant differences were also observed between samples of refined pasta apparently similar for the actual parameters used for the assessment of pasta quality. The results indicated that a number of metabolites undergo a transformation during the pasta-making process depending on the processing conditions adopted. The approach used in this work shows the high potential of metabolite profiling for food investigations with regard to process-related transformation, safety, and nutrition.

Coordinated transcriptional regulation of the divinyl ether biosynthetic genes in tobacco by signal molecules related to defense.

In tobacco, 9-divinyl ethers (DVEs) produced by the lipoxygenase NtLOX1 and DVE synthase NtDES1 are important for full resistance to pathogens. In this work, the regulation of NtLOX1 and NtDES1 expression by signal molecules was investigated in LOX1 promoter-reporter transgenic plants and by RT-qPCR. Methyl jasmonate, ACC and elicitor were shown to coordinately trigger the DVE pathway. Induction was strongly attenuated in the presence of salicylic acid, which seems to act as a negative regulator of the 9-DVE biosynthetic enzymes. Our data suggest that, in tobacco, DVE biosynthesis is cross-regulated by jasmonates, and by other hormonal and signal molecules such as ethylene and SA.

Characterization of a divinyl ether biosynthetic pathway specifically associated with pathogenesis in tobacco.

In tobacco (Nicotiana tabacum), an elicitor- and pathogen-induced 9-lipoxygenase (LOX) gene, NtLOX1, is essential for full resistance to pathogens, notably to an incompatible race of Phytophthora parasitica var. nicotianae (Ppn race 0). In this work, we aimed to identify those oxylipins induced during attempted infection by Ppn race 0 and down-regulated in NtLOX1 antisense plants. Here we show that colneleic and colnelenic acids, which significantly inhibit germination of Ppn zoospores, are produced in roots of wild-type plants inoculated with Ppn, but are down-regulated in NtLOX1 antisense plants. A search for a tobacco gene encoding the enzyme involved in the formation of these divinyl ether (DVE) fatty acids resulted in the cloning and characterization of a DVE synthase (DES) clone (NtDES1). NtDES1 is a 9-DES, specifically converting fatty acid 9-hydroperoxides into DVE fatty acids. NtDES1 has the potential to act in combination with NtLOX1 because, in the presence of the two enzymes, linoleic and linolenic acids were converted in vitro into colneleic and colnelenic acids, respectively. In addition, the pattern of NtDES1 gene expression was quite similar to that of NtLOX1. Their transcripts were undetected in healthy tissues from different plant organs, and accumulated locally and transiently after elicitation and fungal infection, but not after wounding. Visualization of NtDES1-yellow fluorescent protein and NtLOX1-cyan fluorescent protein fusion proteins in tobacco leaves indicated that both localize in the cytosol and are excluded from plastids, consistent with the presumed location of the 9-LOX pathway in plants and the lack of transit peptides for NtLOX1 and NtDES1, respectively. Our data suggest that, in tobacco, NtDES1 and NtLOX1 act together and form DVEs in response to pathogen attack and that this class of oxylipins modulates in vivo the outcome of the tobacco-Ppn race 0 interaction.