Fig. 2: Employing PUP-IT to identify proteins in cellulose synthesis.

a, b Volcano plots show differently enriched proteins in the PafA-CC1 and PafA-LTI6B groups from experiments in N. benthamiana (a) and Arabidopsis seedlings (b), based on a two-sided t-test with permutation-based FDR (FDR = 0.05, S0 = 1). Orange dots represent proteins unique to the PafA-CC1 group, maroon dots represent high-abundance proteins, gray dots represent proteins with no significant differences, and blue dots represent proteins more abundant in the control. CSC and BEN1-related proteins are marked with larger dots. c Co-localization of BEN1-mCherry and EGFP-CC1 in stable transgenic Arabidopsis. Scale bar = 10 µm. d Fluorescence intensity along the transect in (c). Images were taken from cells in the root tip area of 5-day-old seedlings. e BEN1-mCherry was labeled with FLAG when co-expressed with FLAG-Pup(E) and PafA-CC1 in N. benthamiana. RFP trap was used for immunoprecipitation, and α-RFP (mCherry) or α-FLAG antibodies for western blot. p19-only samples served as control. f BEN1-mCherry was co-immunoprecipitated with EGFP-CC1 in N. benthamiana. GFP trap was used for immunoprecipitation, and α-RFP (mCherry) or α-GFP antibodies for western blot. EGFP samples served as the negative control, and p19-only samples as the empty control. Arrowheads point to proteins of interest: BEN1-mCherry (white), EGFP (yellow) and EGFP-CC1 (red). g CC1 is a transmembrane protein with its N terminal facing the cytosol and C terminal in the apoplast, C terminal truncation (∆C), N terminal truncation (∆N). h Membrane split-ubiquitin Y2H was used to detect interactions between BEN1 and CC1, with CC1 constructs designed as shown in (g). Colony growth percentages on selection media from three replicates. Values are mean + SD. Significance was determined by one-way ANOVA followed by a Tukey’s test (p < 0.05). i BiFC assay assessed the interaction between BEN1 and CC1, with CC1 constructs designed as shown in (g). Scale bar = 50 µm. j Relative YFP to RFP ratio was measured along the cell outlines in BiFC images. Values are mean ± SD. Significance was determined by one-way ANOVA followed by a Tukey’s test (p < 0.05, n = 15).