NPR1 is required for root colonization and the establishment of a mutualistic symbiosis between the beneficial bacterium Rhizobium radiobacter and barley.

Non-expressor of pathogenesis-related genes 1 (NPR1) is a key regulator of plant innate immunity and systemic disease resistance. The model for NPR1 function is based on experimental evidence obtained largely from dicots; however, this model does not fit all aspects of Poaceae family, which includes major crops such as wheat, rice and barley. In addition, there is little scientific data on NPR1's role in mutualistic symbioses. We assessed barley (Hordeum vulgare) HvNPR1 requirement during the establishment of mutualistic symbiosis between barley and beneficial Alphaproteobacterium Rhizobium radiobacter F4 (RrF4). Upon RrF4 root-inoculation, barley NPR1-knockdown (KD-hvnpr1) plants lost the typical spatiotemporal colonization pattern and supported less bacterial multiplication. Following RrF4 colonization, expression of salicylic acid marker genes were strongly enhanced in wild-type roots; whereas in comparison, KD-hvnpr1 roots exhibited little to no induction. Both basal and RrF4-induced root-initiated systemic resistance against virulent Blumeria graminis were impaired in leaves of KD-hvnpr1. Besides these immune-related differences, KD-hvnpr1 plants displayed higher root and shoot biomass than WT. However, RrF4-mediated growth promotion was largely compromised in KD-hvnpr1. Our results demonstrate a critical role for HvNPR1 in establishing a mutualistic symbiosis between a beneficial bacterium and a cereal crop.

A scalable downstream process for the purification of the cell culture-derived Orf virus for human or veterinary applications.

The large demand for safe and efficient viral vector-based vaccines and gene therapies against both inherited and acquired diseases accelerates the development of viral vectors. One outstanding example, the Orf virus, has a wide range of applications, a superior efficacy and an excellent safety profile combined with a reduced pathogenicity compared to other viral vectors. However, besides these favorable attributes, an efficient and scalable downstream process still needs to be developed. Recently, we screened potential chromatographic stationary phases for Orf virus purification. Based on these previous accomplishments, we developed a complete downstream process for the cell culture-derived Orf virus. The described process comprises a membrane-based clarification step, a nuclease treatment, steric exclusion chromatography, and a secondary chromatographic purification step using Capto® Core 700 resin. The applicability of this process to a variety of diverse Orf virus vectors was shown, testing two different genotypes. These studies render the possibility to apply the developed downstream scheme for both genotypes, and lead to overall virus yields of about 64 %, with step recoveries of >70 % for the clarification, and >90 % for the chromatography train. Protein concentrations of the final product are below the detection limits, and the final DNA concentration of about 1 ng per 1E + 06 infective virus units resembles a total DNA depletion of 96-98 %.

Selective release of circRNAs in platelet-derived extracellular vesicles.

Circular RNAs (circRNAs) are a novel class of noncoding RNAs present in all eukaryotic cells investigated so far and generated by a special mode of alternative splicing of pre-mRNAs. Thereby, single exons, or multiple adjacent and spliced exons, are released in a circular form. CircRNAs are cell-type specifically expressed, are unusually stable, and can be found in various body fluids such as blood and saliva. Here we analysed circRNAs and the corresponding linear splice isoforms from human platelets, where circRNAs are particularly abundant, compared with other hematopoietic cell types. In addition, we isolated extracellular vesicles from purified and in vitro activated human platelets, using density-gradient centrifugation, followed by RNA-seq analysis for circRNA detection. We could demonstrate that circRNAs are packaged and released within both types of vesicles (microvesicles and exosomes) derived from platelets. Interestingly, we observed a selective release of circRNAs into the vesicles, suggesting a specific sorting mechanism. In sum, circRNAs represent yet another class of extracellular RNAs that circulate in the body and may be involved in signalling pathways. Since platelets are essential for central physiological processes such as haemostasis, wound healing, inflammation and cancer metastasis, these findings should greatly extend the potential of circRNAs as prognostic and diagnostic biomarkers.