Tracking of plasmodesmata localization with the use of fluorescently labelled VAP27 protein

Abstract

Plasmodesmata play a crucial role in plant development processes, biotic and abiotic stress responses. The thick space inside of a plasmodesma between the plasma membrane and desmotubule is tethered by spoke-like elements. VAP27 is a transmembrane protein localized in plasmodesmata that acts as their tether. The aim of the study was to investigate the integration of fluorescently labelled VAP27 protein into plasma membrane in Nicotiana benthamiana plasmodesmata which would enable the tracking of N. benthamiana plasmodesmata localization in vivo. The research was done by infiltration of N. benthamiana with the Agrobacterium tumefaciens cells. A. tumefaciens were transformed with the GreenGate assembled plasmid containing the gene coding for Nterminally labelled VAP27 with EGFP. The EGFP fluorescence was detected by confocal imaging of N. benthamiana leaves. Prior to confocal imaging, the leaves were infiltrated with aniline blue, which acted as plasmodesmata marker. The findings revealed the colocalization of positions of VAP27-EGFP expression and plasmodesmata. The conclusion was made that the integration of VAP27-EGFP into the plasma membrane in plasmodesmata was successful.