Investigating the Effects of Salicylic Acid on Intercellular Trafficking via Plasmodesmata

Plasmodesmata (PD) are plasma membrane-lined pores in plant cell walls that allow trafficking of small molecules and macromolecules, including metabolites, signaling molecules, and some hormones, from one plant cell to another. Interestingly, the plant hormone salicylic acid (SA) does not traffic via PD, but instead moves through the apoplast, but it can function to influence PD trafficking. Indeed, SA is a critical hormonal signal that regulates this cell-to-cell permeability in response to pathogen infection. Previous studies have shown SA to regulate plasmodesmata through regulation of callose, which can occlude PD and limit transport. This study focuses on investigating the cellular mechanisms of how SA could directly or indirectly regulate intercellular trafficking by adjusting PD permeability. Here, we present approaches to investigate how SA is connected to PD transport in epidermal leaf tissues of N. benthamiana. We seek to understand how SA regulates PD through callose accumulation or decumulation at PD in a time and concentration dependent manner. We addressed what callose dynamics are associated with SA-mediated changes to intercellular trafficking by investigating the correlation between callose deposition and PD movement using callose staining with aniline blue and an intercellular GFP transport assay. We also measured PD distribution in SA-treated tissue using a fluorescent PD marker. SA regulates intercellular trafficking via PD by adjusting callose at PD. Short treatment (1hr SA treatment) with SA show reduces cell-to-cell movement. Long treatment with SA reduces intercellular trafficking and, unexpectedly, we observed an increase in cell-to-cell trafficking at 4 hours of SA treatment. Method 1, which required fixed tissues was used to accurately quantify callose dynamics that are associated with SA-mediated changes to intercellular trafficking. We observed strong correlations between PD relative permeability and callose deposition. We also found that SA does not regulate PD formation at the times tested. The results that SA exerts complex regulatory control over PD-mediated intercellular trafficking through callose dynamics.