LLEWELYN, ALEXANDER,JAMES (2020) Investigating the Hydrodynamics of Potato NBLRR Rx1 Using Fluorescence Anisotropy. Doctoral thesis, Durham University.
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The NBLRR family of proteins forms a vital component of the plant immune system but have poorly characterised activity and signalling. Potato NBLRR protein Rx1 confers extreme resistance to Potato Virus X and is known to exhibit DNA binding behaviour. Fluorescence anisotropy is a phenomenon characterised by the unequal emission of light by fluorophores along its axes of polarisation. This property can be exploited using polarised laser light to extract information about rotational velocity and hydrodynamic size. This thesis presents work to construct a time-resolved fluorescence anisotropy microscope system to study Rx1 in vivo, as well as a new method of analysing
the data as a distribution of lifetimes. Lumazine Protein (LumP) was shown to be a suitable protein for these kinds of experiments, with superior properties compared to GFP. Additionally, LumP was found to act as a solubilisation factor for Rx1 in vitro. Anisotropy measurements using a LumP fusion demonstrated an increased size and wider distribution of sizes for Rx1 CCNBARC when bound to ATP compared to ADP. NbGlk binding previously observed was confirmed by this independent method, while NbDBCP was shown to abolish this binding. The solubilised LumP fusion was also shown to restore some phosphatase activity not observed in the refolded protein. Several putative protein interactors were investigated using VIGS, and EIL5
was found to be a promising hit, with silencing shown to enhance Rx1-induced immunity. Plant NBLRR NRC1 was shown to move to DNA in response to Rx1 in planta. Putative SUMO binding site K506R in Rx1 was shown to be essential for Rx1 function. Rx1-LumP was shown to be functional in vivo, although no LumP fluorescence was observed.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Keywords:||Fluorescence, anisotropy, Rx1, NBLRR, Potato, plant, immunity, biochemistry, molecular biology, LumP, hydrodynamics, protein,|
|Faculty and Department:||Faculty of Science > Biological and Biomedical Sciences, School of|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||25 Jan 2021 13:17|