Fig. 6: GRF1 PARylation was required for heterodimer formation with GRF2.

a–c The GRF1 or GRF2 protein performed as dimers in gel-filtration assay. The purified recombinant protein of 6×His-GRF1 or 6×His-GRF2 was filtrated in a size-exclusion column with 1× PBS buffer. The chromatographic showed two elution peaks of indicated proteins corresponding to monomer and dimer, respectively. Milliabsorbance units (mAU). d The PARylation sites mutant GRF1^m3-5 impaired the interaction with GRF2 in vitro. 6×His-GRF1 or 6×His-GRF1^m3-5 was incubated with GST-GRF2 to perform pull-down assays, and the input and pull-down were detected using α-His antibody. e The GRF1^m3-5 mutant showed compromised interaction with GRF2 in appressoria of M. oryzae. Total proteins were extracted and subsequently incubated with α-GFP beads. The total and precipitated proteins were examined by immunoblot using α-GFP or α-FLAG, respectively. Data are representatives of three (a–c, e) or two (d) independent experiments with similar results.