Biochemical and microscopical investigation of heavy metal associated isoprenylated plant protein 7 (HIPP7) as a component and regulator of plasmodesmata

The plant cell wall is a barrier to intercellular, cytoplasmic connectivity. Plasmodesmata (PD) are membrane-lined channels which form specialised gaps across the cell wall to provide and mediate cell-to-cell connectivity. This controlled connectivity allows for the sharing of resources and signals to coordinate the efforts of the various cell types. Whilst the overall structure of plasmodesmata has been described in significant detail, only recent years have seen notable progress in proteomic identification of their molecular components. Conversely, the plasmodesmata-specific functions of newly identified plasmodesmata-localised proteins remain to be characterised. The mechanisms for plasmodesmata regulation are not fully understood and do not reach the level of sophistication expected of a system so essential to plant life. One protein family for which multiple members have been shown to be plasmodesmata-associated are Heavy Metal Associated Isoprenylated Plant Proteins (HIPPs), a group of plant-specific metal chaperones. However, there was previously no information regarding their plasmodesmata-specific functions. In this thesis, HIPP7 is introduced as a novel regulator of cell connectivity. The effect of HIPP7 expression on cell-to-cell trafficking of fluorescent tracers, virus spread, accumulation of the PD-regulating cell wall component callose, and the localisation of other known components of PD is characterised. Serial Block Face Scanning Electron Microscopy (SBF-SEM) from transverse sectioning in the root meristem was used to uncover effects on plasmodesmata density and architecture and a biotin ligase system for proximity labelling has produced a HIPP7 interactome. These varied, targeted investigations of HIPP7 have provided further insight into the regulation of plasmodesmata and summarise numerous methods for investigation of a protein otherwise largely uncharacterised as a PD component.