Fig. 5

OsFBK16 interacts with and degrades OsPAL1 to modulate rice blast resistance. a The AH109 yeast strain containing BD-OsPAL1 mated with the Y187 strain containing the E3 ubiquitin ligase library for screening. DDO, SD-Leu-Trp, QDO, SD-Leu-Trp-His-Ade. b Co-IP assay of OsPAL1 with OsFBK16 in vivo. N. benthamiana leaves were agro-infiltrated with the indicated plasmid combinations. Total protein was extracted and immunoprecipitated with anti-HA/Agarose beads. Immunoblot detection was performed using anti-GFP or HA antibody as indicated. c Degradation assay of OsPAL1 by OsFBK16 in the presence or absence of 50 μM MG132. N. benthamiana leaves were agro-infiltrated with OsPAL1-GFP and OsFBK16-HA or GUS-HA. Samples were collected, and total protein was extracted. The protein abundance of OsPAL1 was detected by immunoblotting using an anti-GFP antibody, and OsFBK16 and GUS were detected using an anti-HA antibody. ACTIN was used as the internal control. OsPAL1 transcript levels were measured by RT-PCR, and ACTIN was used as the internal control. d–f Disease symptoms of two representative OsPAL1-GFP overexpression lines and NPB seedlings after punch inoculation with the compatible M. oryzae isolate RB22. d The images were taken 14 days after inoculation. e The disease lesion area was measured with the ImageJ software. An asterisk indicates a significant difference between WT and OsPAL1-GFP overexpression plants according to Student’s t test (**p < 0.01). f Relative fungal biomass was calculated by measuring the expression of the M. oryzae MoPot2 with the DNA-based quantitative PCR assay. An asterisk indicates a significant difference between WT and OsPAL1-GFP overexpression plants according to Student’s t test (**p < 0.01). g–i Disease symptoms of the osfbk16 mutant and NPB seedlings after punch inoculation with RB22. g The images were taken 14 days after inoculation. h The disease lesion area was measured with the ImageJ software. An asterisk indicates a significant difference between WT and the osfbk16 mutant according to Student’s t test (**p < 0.01). i Relative fungal biomass was calculated by measuring the expression of the M. oryzae MoPot2 with the DNA-based quantitative PCR assay. An asterisk indicates a significant difference between WT and osfbk16 mutants according to Student’s t test (**p < 0.01)