Investigating the PrpS-PrsS (pollen and pistil SI determinant) interaction

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This project will examine the nature of PrpS and its proposed "receptor-ligand" type interaction with PrsS, using a range of live-cell approaches. We will use a range of live-cell approaches to study the SI determinant-ligand interactions in transgenic Arabidopsis pollen protoplasts, transfected animal cells and poppy pollen tubes transiently expressing PrpS-GFP using a biolistic approach. We will also use Alexa-tagged PrsS for some experiments. This will provide a detailed determination of the nature, localization, distribution and dynamics of the receptor-ligand interaction. (A) aims to establish the nature of PrpS as an ion channel We will express PrpS in a heterologous cell system (Xenopus oocytes and /or HEK cells) to build on preliminary data to firmly establish that PrsS functions as an ion channel. Patch- clamping will allow us to identify PrsS-induced activation and kinetics, channel conductance and selectivity and pharmacological properties. (B) and (C) focus on studying PrpS dynamics and interactions with PrsS and identifying amino acids/domains involved in recognition/function. We will use live-cell imaging using fluorescence microscopy, including: Bimolecular fluorescence complementation (BiFC), Total internal reflection fluorescence microscopy (TIRF-M), FRAP and FRET to study PrpS-PrsS interactions in detail. Site-directed mutagenesis on the predicted extracellular 35 aa loop and also making PrpS chimeras by domain-swopping this region between allelic variants (e.g. replace S1 with S3) will establish which changes result in loss or altered function or S-specificity. These approaches will allow us to establish if PrpS forms a multimeric complex, establish the subcellular localization/activity of PrpS/PrsS, examine probe real-time PrpS-PrsS interaction dynamics in live cells, and establish the nature of the allele-specificity of the PrpS-PrsS interaction.

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