Fig. 6: OsBHT is a putative substrate of OsPUX8B.2–OsCDC48-6.

a Co-IP assay showing that OsPUX8B.2 associates with OsBHT but not with OseIF5A or OsSTI. b Co-IP assay showing that OsBHT only binds to OsPUX8B.2 but not to OsPUX8B.1 or OsPUX8B.3. c Co-IP assay showing that the association between OsBHT and OsPUX8B.2 requires the UBA and UBX domains of OsPUX8B.2. d Colocalization of OsBHT-mCherry with free GFP (upper panel) or OsPUX8B.2-GFP (lower panel). Fluorescence intensities (gray values) across the white lines are shown in the line plots on the right. e In vivo ubiquitination assay showing that OsBHT is ubiquitinated in rice protoplasts transfected with the indicated constructs. f Immunoblot analysis of proteins extracted from rice protoplasts showing that OsPUX8B.2–OsCDC48-6 promotes the turnover of OsBHT. Numbers below the protein bands indicate the relative abundances of OsBHT-cMyc compared to β-tubulin. g, h Competitive Co-IP (g) and SLC (h) assays showing that MoNLE1 disrupts the association between OsBHT and OsPUX8B.2. i, j Phenotypes of Osbht mutants (i) and OsBHT-GFP-OE plants (j) infected with blast fungus. Punch inoculation was performed with blast isolate RB22, and the disease symptoms were recorded at 10 dpi. Lesion area and relative fungal biomass were used to quantify disease severity. Scale bars, 1 cm. Data were shown as boxplot of the number of indicated biological replicates displaying the maximum and minimum, first and third quantiles, and the median (h); or means ± s.d. from the indicated number of independent biological replicates (i, j). Data were analyzed by two-tailed Student’s t-tests (h) or one-way ANOVA followed by Tukey’s test (i, j). Exact p (h) or adjusted p (i, j) values were shown in figures. Data in a–g are representatives of three independent experiments with similar results.