Analysis of the Spatiotemporal Organization of Immune Receptors in Plant Plasma Membrane by sptPALM and the Impact of HIR2 as Putative Organizing Factor

Abstract

In plants, the recognition of external and internal stimuli is mediated by plasma membrane-localized receptors, which are heterogeneously distributed and organized in nanodomains. How their formation is regulated and how the specificity of multiprotein receptor complexes is determined, remains unclear. So far, the formation and the maintenance of multiprotein receptor complexes is largely unknown, though it turns out that the spatiotemporal organization and the nanoscale dynamics of membrane proteins is a fundamental aspect of the function of signaling processes. In this work, the function and the involvement of HIR2, a SPFH-domain containing membrane localized protein, which is hypothesized to be involved in the regulation of complex assembly of immune receptor kinases at the plasma membrane, was investigated by genetic and biochemical approaches. The direct interaction of HIR2 with the BIR2 and BIR3 was confirmed by multiple independent interaction experiments. Also, it was demonstrated that HIR2 directly interacts with the co-receptor BAK1, as well as with the PRRs FLS2 and CERK1. Besides the interactions, it remains to be elucidated how HIR2 is involved in PTI signaling. Additionally, HIR2 is suggested to be involved in developmental signaling pathways since it is associated with BRI1, and the hir2-5 mutant exhibits a dwarf growth phenotype, similar to weak bri1 alleles. To study the nanoscale dynamics and cluster formation of plasma membrane localized proteins, single and dual color sptPALM were successfully established for their application in planta and the nanodomain formation was computed by Voronoi tessellation-based segmentation. The analysis revealed a heterogeneous distribution and cluster formation of all studied proteins within the plasma membrane. The receptor kinases FLS2 and BIR3 possessed low lateral mobility within the plasma membrane, which was unaffected by flg22 treatment. In contrast, BAK1 occurred in two different populations with distinguishable lateral mobility behaviors, and flg22 treatment resulted in a reduced mobility of BAK1. Thus, it is hypothesized that BAK1 dynamically associates with different nanodomains and it is not permanently present in a preformed complex with FLS2. With this work, new insights were given (i) into the impact of HIR2 as a putative organizing factor for the assembly of spatially and temporally stable multiprotein complexes at the plasma membrane and (ii) into the spatiotemporal organization of immune receptor kinases and their modulation by ligand perception.