An evolutionarily conserved motif is required for Plasmodesmata-located protein 5 to regulate cell-to-cell movement

摘要

a A receptor-like domain structure of a PDLP protein. SP, signal peptide; ExD, extracellular domain; TMD, transmembrane domain; CT, C-terminal cytosolic tail. b The TMD sequence of PDLP5 and a sequence logo spanning the TMD region of 491 PDLPs, generated using WebLogo. Conserved alanine and glycine residues are bold typed. c–e. TMV-GFP viral movement assays. For each movement experiment set, at least ten plants were used for mock and test treatments, and each set is repeated at least twice. A schematic illustration of the assay (c) and photographs of N. benthamiana plants exhibiting the differential impact of PDLP5 or PDLP5VV, compared with mock, on the systemic movement of TMV-GFP 5-days post agroinfiltration (dpa) (d). The levels of PDLP5 and PDLP5VV transcripts were determined by semi-quantitative RT-PCR using N. benthamiana leaves harvested at 3-dpa. Three biological and two technical repeats were performed per treatment. Transcript levels were normalized to that of elf1-α (e). Asterisks, significant differences analyzed with a one-way ANOVA followed by Fisher’s Least Significant Differences (LSD) test at the probability of P < 0.001 (see Source Data for details). Error bars, standard deviation. f–h Cell-to-cell movement assays using free mCherry as a reporter. Representative confocal images show the extent of the mCherry movement. Asterisks, transfected cells (f). Quantification of the total (g) and the extent (h) of mCherry movement. Over 550 transfected cells were examined in total using at least ten plants per treatment in three independent experimental repeats (n = 3). “Total” represents the percentage of the total number of cells exhibiting mCherry movement out of the total number of transfected cells (g), while “extent” represents the percentage of cells exhibiting movement into one or more neighboring cells out of the total number of transfected cells (h). Statistical analysis, a one-way ANOVA analysis followed by LSD test at the probability of P < 0.05. Letters indicate significantly different groups. Error bars, standard deviation. i Representative confocal images of PDLP5- or PDLP5VV-EGFP (false-colored in yellow) showing their localization at plasmodesmata (arrows) along the cell junctions of N. benthamiana epidermal cells as shown by co-localization with aniline-blue-stained callose (false-colored in magenta).