Transient expression of poliovirus-like particles in plants. Developing a synthetic polio vaccine

Plants, or cell suspension cultures derived from them, are a promising platform for the production of biologics and pharmaceuticals. In this work transient expression utilising the pEAQ vector system was deployed for the expression of virus-like particles (VLPs) in Nicotiana benthamiana or N. tabacum BY-2 cell suspension cultures. The results presented in this thesis demonstrate the potential of plant systems for the production of VLP-based vaccines. VLPs of the fish virus, Nervous necrosis virus (NNV), were successfully produced in plants by transient expression of the coat protein. The protein self-assembled into T = 3 particles, which appeared to be morphologically identical to the wild-type NNV when analysed by high resolution microscopy but were devoid of nucleic acid. In addition, transgenic BY-2 cell suspension lines were generated expressing correctly assembled NNV VLPs. Poliovirus (PV)-like particles from all three PV serotypes, containing either the wt coat protein or coat proteins with stabilising mutations, were successfully expressed in plants. These were generated by co-expression of the structural polyprotein P1 and the proteinase 3CD. Sufficient quantities of purified particles could be obtained for structural and immunological analysis. Mice carrying the gene for the human PV receptor were protected from wild-type PV when immunised with the plant-made stabilised PV VLPs. Structural analysis of the stabilised mutant of PV3 at 3.6 Å resolution revealed a structure almost indistinguishable from wild-type PV3, with the stabilising mutations having no effective on the antigenic surface of the particle. To make the product more attractive to the vaccine industry, tobacco BY-2 cells have been successfully tested for the transient expression of the above-mentioned PV mutant VLPs using the cell-pack method

Ups and downs of a transcriptional landscape shape iron deficiency associated chlorosis of the maize inbreds B73 and Mo17

BACKGROUND: Improving nutrient homeostasis is a major challenge of a sustainable maize cultivation, and cornerstone to ensure food supply for a growing world population. Although, iron constitutes an important nutrient, iron availability is limited. In this respect, iron deficiency associated chlorosis causes severe yield losses every year. Natural variation of the latter trait has yet not been addressed in maize and was therefore studied in the present analysis. RESULTS: In this study, we i) report about the contrasting chlorosis phenotypes of the inbreds B73 and Mo17 at 10 and 300 μM iron regime, ii) identified over 400 significantly regulated transcripts (FDR < 0.05) within both inbreds at these growth conditions by deep RNA-Sequencing, iii) linked the gained knowledge with QTL information about iron deficiency related traits within the maize intermated B73 by Mo17 (IBM) population, and iv) highlighted contributing molecular pathways. In this respect, several genes within methionine salvage pathway and phytosiderophore synthesis were found to present constitutively high expression in Mo17, even under sufficient iron supply. Moreover, the same expression pattern could be observed for two putative bHLH transcription factors. In addition, a number of differentially expressed genes showed a co-localisation with QTL confidence intervals for iron deficiency related traits within the IBM population. CONCLUSIONS: Our study highlights differential iron deficiency associated chlorosis between B73 and Mo17 and represents a valuable resource for differentially expressed genes upon iron limitation and chlorosis response. Besides identifying two putative bHLH transcription factors, we propose that methionine salvage pathway and sterol metabolism amongst others; underlie the contrasting iron deficiency related chlorosis phenotype of both inbreds. Altogether, this study emphasizes a contribution of selected genes and pathways on natural trait variation within the IBM population