Fig. 2
HaRxL103Emoy2 is recognized by RPP4 in an EDS1-dependent manner. a N. benthamiana leaves were inoculated with Agrobacterium containing estradiol-inducible GFP-HaRxL103Emoy2 (Est-103Emoy2), RPP4-FLAG and GUS-RNAi or NbEDS1-RNAi constructs. The different inoculation sites were infiltrated with 20 μM estradiol (Est) or water (Mock) 24 h after the inoculation. The leaves were photographed under UV at 2 days after the infiltration. b Confirmation of proteins accumulation and NbEDS1 silencing. Total proteins and RNAs were prepared from N. benthamiana leaves described above 8 h after infiltration with estradiol or water. Immunoblot analyses were done using anti-GFP (top panel) and anti-FLAG (middle panel) antibodies. Protein loads were monitored by Coomassie Brilliant Blue (CBB) staining of the bands corresponding to ribulose-1,5-bisphosphate carboxylase (Rubisco) large subunit (bottom panel). The bar chart indicates expression levels of NbEDS1 determined by qRT-PCR. Data are means ± SDs from three technical replicates. The experiments were repeated two times with similar results. c In planta interaction between HaRxL103Emoy2 and RPP4. Co-immunoprecipitation was performed with extracts from N. benthamiana leaves co-expressing GFP or GFP-HaRxL103Emoy2 with RPP4-FLAG. MACS MicroBeads with GFP antibody were used for immunoprecipitation, and anti-GFP (upper panel) and anti-FLAG (lower panel) antibodies were used to detect the related proteins in the immunoprecipitates. Protein accumulation (d), AtPR1 expression (e), and Hpa growth (f) in Arabidopsis Col-0 and Col-0 rpp4 transgenic lines containing estradiol-inducible GFP (Est-GFP) and Est-103Emoy2 constructs. d Total proteins and RNAs were prepared from 2-week old plants 24 h after spray treatment with 40 μM estradiol or water. Immunoblot analyses were done using anti-GFP as described in (b). Asterisks indicate the detected GFP or GFP-HaRxL103Emoy2 constructs. e The expression level of AtPR1 was determined by qRT-PCR. Data are means ± SDs from three biological replicates. Different letters indicate significantly different values at p < 0.05 (one-way ANOVA, Tukey’s HSD). f Three-week-old transgenic lines 24 h after spray treatment with estradiol or water were inoculated with Hpa Waco9. Conidiospores were harvested and counted at 5 dpi. Data are means ± SEs from five biological replicates. Different letters indicate significantly different values at p < 0.01 (one-way ANOVA, Tukey’s HSD)
