Quantification of Protein Enrichment at Plasmodesmata

Background

Currently, confirmation of protein localization to PD by confocal imaging is based primarily on two different approaches. On the one hand, the molecular composition of the cell wall surrounding PD differs. While the cell wall is highly enriched in cellulose, the environment near the PD is enriched in callose, a beta (1-3) glucan polymer that can easily and specifically be stained with fluorophore Aniline Blue. This staining presents the considerable advantage of being used as a PD marker without crossed lines. On the other hand, proteomic studies have identified specific PD proteins and led to their characterization (Faulkner et al., 2005; Fernandez-Calvino et al., 2011; Grison et al., 2015; Brault et al., 2019). These proteins can be used as PD markers in subsequent studies when tagged with fluorescent proteins in transient or stable expression in plants (Thomas et al., 2008; Simpson et al., 2009). Note that PD proteins can be exclusively associated with PD but can also present a dual localization within different cellular compartments as for Synaptotagmin1 (SYT1), Multiple C2 domains and Transmembrane regions Protein 4 (MCTP4) which associate with both the Endoplasmic Reticulum and PD (Levy et al., 2015; Brault et al., 2019). Since PD are dynamic structures responding to developmental and environmental cues, their molecular constituents may vary conditionally (Benitez-Alfonso et al., 2013; Stahl et al., 2013; Han et al., 2014; Sager and Lee, 2014; Otero et al., 2016; Stahl and Faulkner, 2016). Thus, Receptor Like Kinases (RLKs), such as the Leucine-Rich-Repeat RLKs Qian Shou Kinase 1 (QSK1) and Inflorescence Meristem Kinase 2 (IMK2) or the Cystein-Rich Receptor Kinase 2 (CRK2), are able to dynamically relocate to PD upon osmotic stress conditions (Grison et al., 2019; Hunter et al., 2019). The study of the dynamic localization of protein in vivo requires the development of quantification methodologies. Using confocal microscopy, we developed a ratiometric calculations to evaluate the PD enrichment of a given protein using PD markers, hereinafter referred as “PD Index”. The PD Index can be used for co-localization experiments but also as reference points for characterizing mutants, drugs or growing conditions that could modify the degree of proteins PD association (Perraki et al., 2018; Brault et al., 2019; Grison et al., 2019).