Implementing In Vitro and In Vivo Approaches to Analyze Lipid-Protein Interactions in Growing Tobacco Pollen Tubes

The spatial organization of anionic phospholipids such as PI4P, PI(4,5)P₂, phosphatidic acid, and phosphatidylserine is a defining feature of plasma membrane domains that support the extreme polarity of pollen tube growth. Many key protein regulators-including exocyst subunits, ROP pathway components, and cytoskeletal effectors-associate with the membrane through lipid-dependent mechanisms. This chapter provides comprehensive protocols for analyzing these interactions using complementary in vitro and in vivo approaches. We describe the production of tagged plant proteins in Escherichia coli and wheat-germ extracts, followed by assessment of lipid-binding specificity using protein-lipid overlays and quantitative liposome co-sedimentation assays. To test the physiological relevance of these interactions, we detail methods for transient transformation of tobacco pollen via biolistics, followed by microscopic colocalization of proteins of interest with genetically encoded fluorescent lipid reporters, and changes in their membrane recruitment upon overexpression of lipid-modifying enzymes. The integrated workflow enables multifaceted characterization of lipid-protein interactions in their native context and provides a versatile platform for studying membrane identity, signaling, and vesicle trafficking in plant tip-growing cells.