To begin, obtain Nicotiana benthamiana plant infiltrated with the Agrobacterium tumefaciens cells harboring desired vectors. For protein localization studies, harvest two leaves from two plants at different time points after the infiltration. Using a blade cut the infiltration zone into slices away from the vein.
Place the tissue sample with the abaxial side up on a drop of sterile water at the center of a microscope slide. Cover the slide with a cover glass ensuring no bubbles are trapped. Visualize the fluorescent signal of autofluorescent tags in the infiltrated area with a laser scanning confocal microscope.
Collect 20 images for each condition using at least two independent biological replicates. Score plasmodesmal localization of the tested protein based on the diagnostic punctate appearance of the signal at the cell periphery. TVCV MP-EGFP and PDL5-EGFP proteins demonstrated clear punctate localization to plasmodesmata.
While PDCB1-EGFP showed localization to both plasmodesmata and endoplasmic reticulum on day one, post infiltration. Both MP and PDLP5 began accumulating at plasmodesmata on day one post infiltration, reaching maximum intensity on day two and remained stable for five days. PDCB1 exhibited strong endoplasmic reticulum localization until day three, after which plasmodesmal localization became visible in fewer cells.
