Abstract
Plasmodesmata (PD) are intercellular channels between walled plant cells that enable the transportation of materials between adjacent cells, which are important for plant growth and development. The permeability of PD must be tightly regulated. Assays to determine the permeability of PD are crucial for related studies on the regulation of PD development and permeability. Here we describe an assay for the determination of PD permeability via the observation and quantification of GFP diffusion and cell-to-cell transport of CMV MP-GFP in Arabidopsis leaves.
Keywords: Plasmodesmata, PD permeability, GFP, CMV MP-GFP, Particle bombardment
Background
Plasmodesmata (PD) are plant-specific channels between cells, which are important for plant growth and development as well as the interaction between plants and the surrounding environment (Maule et al., 2011; Lee, 2015; Cheval and Faulkner, 2018). Water and small molecules can pass through PD freely. Other macromolecules, e.g., proteins, RNA, and some pathogens, can also move via PD. However, the mobility of those molecules between adjacent cells is tightly regulated, although the mechanism underlying this transport is not well understood (Sevilem et al., 2015). Currently, there are several approaches reported for measuring the permeability of PD in plant tissues. One of the methods is to visualize the diffusion of fluorescent dextrans or other fluorescent probes from the targeted cells into neighboring cells (Ding et al., 1996). This approach allows the researchers to select fluorescent dextrans of different sizes which will facilitate the study of PD permeability. In addition, this method also allows researchers to perform the coinjection of fluorescent dextrans with special drugs or proteins that allows the colleagues to assay the effect of those drugs or proteins on the permeability of PD. However, this method requires a rigorous experimental system and a highly experienced operator to perform the experiments. The second method is Drop-ANd-See (DANS) which uses the 5(6)-carboxy fluorescein diacetate (CFDA) for the rapid assessment of PD permeability (Cui et al., 2015). DANS is a simple and rapid approach, but it is impossible to trace the spread of CFDA at the single cell resolution. The researchers in this field also used the green fluorescent protein (GFP) (Crawford and Zambryski, 2001; Liarzi and Epel, 2005) and Cucumber mosaic virus (CMV) Movement Protein (MP)-GFP (CMV MP-GFP) (Iglesias and Meins, 2000) as probes to assay the permeability of PD. The expression of GFP or CMV MP-GFP was achieved by delivering plasmids expressing them into plant cells via particle bombardment. This method enables the researchers to visualize the diffusion of GFP from targeted cells into adjacent cells or cell-to-cell movement of CMV MP-GFP. Here, we introduce the detailed method of GFP diffusion and cell-to-cell movement of CMV MP-GFP, which is adapted from our recently published paper (Diao et al., 2018).
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Acknowledgments
This work was supported by grants from national natural science foundation of China (31471266; 31671390).
Competing interests
The authors declared that they have no conflicts of interest to this work.
References
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