Argonautes are conserved RNA-binding proteins that can regulate gene expression post transcriptionally through a process known as RNA interference (RNAi). This is done through the use of small RNAs, e.g. sRNAs that act as a guide for the argonautes, allowing for sequence-specific binding to the target site. This interaction has been studied in many organisms, one of which is the model organism Dictyostelium discoideum. D. discoideum is an amoeba that has been used extensively in genetic experiments due to its unique lifestyle, and ease of use. Being a eukaryotic, unicellular organism, it proves to be a great tool for the study of regulatory systems in eukaryotes, allowing us to study this argonaute-sRNA interaction in detail. By analysing which RNAs bind to the argonautes, we can better understand which genes these proteins regulate and what role RNAi has in the organisms as a whole.  In this study, I investigate three of the five argonautes found in D. discoideum, namely agnA, agnC and agnE. By transforming FLAG-tagged versions of these genes into the amoeba, I successfully express two of these modified proteins in D. discoideum and verified expression by using antibodies designed specifically to recognise the FLAG-tags. This opens up the possibility for the characterisation of the argonaute proteins to better understand their role and function in the regulation of genes. / <p>The Biology Education Centre (IBG) is the responsible department. </p><p></p><p>Presentation has been made through Zoom. </p>

Wheat is one of the most essential food sources in the world. However, there has been serious yield loss of wheat production due to stripe rust disease caused by the fungal pathogen Puccinia striiformis f. sp. tritici. The cost-effective and long-lasting defense to the disease can be achieved by generating genetically resistant crops against the disease forming pathogens. To accomplish this, first step is to acquire knowledge in the plant pathogen interactions of the crop and the pathogen of interests at the cellular and the molecular level. In this thesis research, PstHa2a5 candidate effector gene from Puccinia striiformis f. sp. tritici is investigated to identify its role and interaction between host factors in yellow rust infected Triticum aestivum L. The gene construct was engineered with FLAG-tag fusion at its N-terminus, and synthesized. This construct was cloned into pJL48-TRBO vector for an expression in Nicotiana benthamiana via agrobacterium-mediated gene transformation. The expressed protein structure with FLAG-tag was purified, and immunoprecipitated with one putative N. benthamiana interactor by immunoprecipitation experiments. This candidate interactor protein will be identified with Mass Spectroscopy. In addition to this, subcellular localization of the effector candidate was examined in N. benthamiana plant. This was achieved by cloning PstHa2a5 gene construct in pK7WGF2 gateway destination vector and localization is determined by GFP expression in N. benthamiana after agrobacterium-mediated gene transformation.