Fig. 5: GRF1 PARylation is required for virulence.

a–c The mutations of the GRF1 PARylation sites substantially diminish its PARylation in vitro. The indicated mutants of GRF1 PARylation sites were incubated with MBP-PARP1-HA in the PARylation buffer with or without NAD⁺, respectively. The PARylation activities were analyzed with α-PAR antibody. The experiments were repeated two (a, b) or three (c) times with similar results. d The PARylation mutants of GRF1 failed to rescue the Δgrf1 virulence defect. The seedlings of rice cultivar CO39 were spray-inoculated with the spore suspensions of the wild type and GRF1 PARylation mutant strains. The leaves with representative symptoms and statistical analysis of lesion numbers at different levels at 1-week post-infection are shown. Values are means ± s.d. from three biological replicates. e The invasive growth and infectious hyphae (IH) of the GRF1 PARylation mutants in barley leaf at 24 hpi. One hundred infectious hyphae were analyzed for each strain. Values are means ± s.d. from three biological replicates. f The incipient collapse rate of the GRF1 PARylation mutants. The percentage of collapsed appressoria was recorded and displayed as boxes with individual data points from three independent repeats. The maximum and minimum, the first and third quantiles, and the median were shown. Significant differences were determined using two-way ANOVA followed by Tukey’s test and marked with different letters.