Fig. 6

Analysis of GSL8 physical association with proteins involved in callose synthesis. a-f BiFC assay showing the interaction between YC fusion of GSL8 and YN fusions of AtBG_PPAP (a), PDLP5 (b), UDPG (c), SCD1 (d) and SUS1 (e). Interactions between GSL8 and AtBG_PPAP/ PDLP5 appear to be localized at the cell membrane and plasmodesmata (white arrowheads) (a-b). GSL8 interacts with UDPG and SUS1 in the cytoplasm and on the ER (c and e). Interaction of GSL8 with SCD1 occurs at the cell membrane and the cell plate (d). Subcellular localization of GSL8-YFP fusion shows its localization at the cell membrane (f). There was no interaction between GSL8-YC and GmIFS2-YN, an ER membrane-localized protein from soybean [70], and pEG100-YN which were used as negative control (g-h). Scale bars = 20 μm. i FRET confirms GSL8 interaction with PDLP5, SCD1, AtBG_PPAP and SUS1. No interaction was found with UDPG and free CFP (data not shown). The boxes signify the upper (dark grey) and lower (light grey) quartiles, and the median is represented by a short black line within the box for each. The upper and lower “whiskers” represent the entire spread of the data. The data used for FRET efficiency calculation and statistical analysis were obtained from three independent experiments and three biological replicates for each experiment. In all cases, values reported are the mean ± SEM. j MYTH assay. Colonies of transformed yeast cells growing on transformation selection media (TSM) and interaction selection media (ISM) indicate successful interaction of bait (GSL8) and prey (AtBG_PPAP, PDLP5, GSL10, SUS1 and SCD1). NubI was used as a positive control. NubG and empty vector were used as negative controls