This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
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Plant membrane immune receptor proteins are essential for initiation of immune responses and therefore their function is vital for plant survival. These receptors typically belong to receptor-like-kinase (RLK) or receptor-like-protein (RLP) protein families. In general, RLKs are comprised of an extracellular, transmembrane and kinase domain. The extracellular detects signs of pathogen invasion, the transmembrane dictates the localisation of the protein and the kinase domain initiates signalling cascades within the cell. RLPs have similar structure to RLKs but lack the intracellular kinase domain hence needing separate co-receptors for signalling. The function of RLKs and RLPs is regulated by a variety of mechanisms – including proteolysis. During proteolysis, a protein cleaving enzyme termed protease cuts a protein generating discrete pieces called proteoforms. This process may either activate or inactivate the protein and could even generate proteoforms with novel functions. RLKs and RLPs can undergo proteolysis but this is wildly understudied in plants.
The aim of my work during the internship was to validate candidates of RLKs proteolysis. Focusing particularly on a form of proteolysis termed shedding in which the protease excises the extracellular domain of the receptor. Prior to my joining the Plant Chemetics lab at the University of Oxford, former PhD student Emma Thomas used Protein Topography and Migration Analysis Platform (PROTOMAP) technology to generate a proteomics dataset of all proteins and their proteoforms in the leaves of model plant Nicotiana benthamiana. Dr Mariana Schuster then mined this proteomics dataset to find suitable receptor candidates that produced stable proteoforms. This datamining yielded 33 candidates for receptor shedding.
My main experimental aim was to validate these 33 shedding candidates in planta using overexpressed GFP c-terminal fusions of the proteins followed by anti-GFP western blots. To do this, the target genes were first inserted into a plasmid backbone containing the sequence of a GFP tag via Gibson assembly and cloned in Escherichia coli. After construct validation, the plasmids were transformed in Agrobacterium tumefaciens prior to agroinfiltration into N. benthamiana plants. This strategy allows for the overexpression of candidate proteins in plants by harnessing the natural genetic engineering capacities of A. tumefaciens. Using this inserted GFP tag as a proxy for successful transient transformation we were able to scan and detect the presence of GFP in infiltrated leaves on a fluorescent scanner.
Imaging was followed by total proteome extraction, proteome separation via SDS Page and anti-GFP western blots. Our western blots indicated there were stable proteoforms corresponding to proteolysis in all candidates that reached the end of the pipeline. We were able to verify this when calculating the molecular weight of the proteoforms – proving that some of these comprise the kinase domain and spanned the transmembrane domain but lacked the extracellular domain (Figure 1). This validates the PROTOMAP predictions of presence of stable proteoforms in planta that were potentially generated by shedding. During my time at the Plant Chemetics lab I was able to validate shedding for three candidates with Dr Schuster identifying one further after I had left. I moreover started the cloning and expression of four further candidates.
My experience in the Plant Chemetics lab has been invaluable for gaining confidence in a lab setting and my time at the University of Oxford has changed my perspective on what a career in research could entail. I feel truly grateful that I could experience this role where I could both discuss other’s research in the lab and receive such support from my supervisor and colleagues. For this I would like to say thank you to the BSPP for funding this research and to Dr Mariana Schuster and colleagues at the Plant Chemetics lab for their supervision and guidance.
University of Bristol
Figure: Diagram of an RLK and its proteoforms. Proteoform A corresponds to the kinase domain whereas proteoform B spans the kinase and transmembrane domain. Proteoform B is therefore potentially generated via a shedding event. SP= signal peptide, TM= transmembrane domain”.