Figure 1

Interaction between BAK1^LRR and BIR1^LRR is required for BIR1 inhibition of BAK1-mediated plant immunity. (A) Gel filtration profiles of BAK1^LRR and BIR1^LRR at pH 6.0. The vertical and horizontal axes represent ultraviolet absorbance (λ = 280 nm) and elution volume (mL), respectively. Bottom panel, coomassie blue staining of the peak fractions following SDS-PAGE. The numbers shown on the top of the SDS-PAGE gels indicate elution volumes (mL). Frame colors of the SDS-PAGE gels are equivalents to those of the gel filtration profiles for proteins indicated. MM: molecular weight maker. Hiload 200 was used for the gel filtration assays. (B) Native-PAGE coomassie blue staining of the peak fractions for the gel filtration at pH 6.0 in (A). (C) Overall structure of the BAK1^LRR-BIR1^LRR complex. “N” and “C” represent the N- and C-terminus, respectively. (D) Detail interactions between BAK1^LRR and BIR1^LRR. The left panel, the interface between the N-terminal side of BIR1^LRR and BAK1^LRR. The right panel, the interface between the C-terminal portion of BAK1^LRR and BIR1^LRR. Red dashed lines indicate polar interactions and their distances are labeled. T, Thr; L, Leu; N, Asn; S, Ser; Y, Tyr; D, Asp; H, His; R, Arg; W, Trp; V, Val; I, Ile; F, Phe. (E) Mutagenesis analysis of the BAK1^LRR-BIR1^LRR (W71A) complex using gel filtration. (F) Mutagenesis analysis of the BAK1^LRR (T190R)-BIR1^LRR complex using gel filtration. (E and F) The assays were performed as described in (A). (G) Morphological phenotypes of transgenic plants expressing the BIR1 (T103Q) -HA or BIR1 (W71A)-HA protein in bir1-1, and the BAK1-HA or BAK1 (T190R)-HA protein under its native promoter in wild type background (Col-0). The photograph shows four-week-old soil-grown plants. Expression of proteins was detected by western blot using an anti-HA antibody. (H and I) Expression levels of PR1 in the indicated genotypes as determined by quantitative RT-PCR. Two-week-old seedlings grown on ½ MS plates were used for the assays. Values were normalized to the expression levels of ACTIN1. The data are shown as means ± SD (n = 3) with one-way ANOVA and Tukey's test. Different letters indicate significant differences (P < 0.01). The experiments were repeated three times with similar results. (J) Growth of H. a. Noco2 on seedlings of the indicated genotypes. The data are shown as mean ± SD (n = 3) with one-way ANOVA and Tukey's test. Different letters indicate significant differences (P < 0.01). The experiments were repeated three times with similar results. (K) Structural superimposition of the BAK1^LRR-BIR1^LRR complex with that of FLS2^LRR-flg22-BAK1^LRR using BAK1^LRR as the template. “N” and “C” represent the N- and C-terminus, respectively. Color codes are indicated. This alignment was performed by the program COOT. (L) FLS2^LRR-flg22 releases BAK1^LRR from the BAK1^LRR-BIR1^LRR complex in gel filtration at pH 6.0. BIR1^LRR and BAK1^LRR with a molar ratio of about 2.5:1 were mixed together and incubated at 4 °C for 30 min. The FLS2^LRR-flg22 complex was then added to the mixture for gel filtration. The molar ratio between FLS2^LRR and BAK1^LRR was about 2:1. Shown in the left panel are gel filtration profiles of proteins indicated. The vertical and horizontal axes represent ultraviolet absorbance (λ = 280 nm) and elution volumes (mL), respectively. Right panel, coomassie blue staining of the peak fractions following SDS-PAGE. The numbers shown on the top of the gels indicate elution volumes (mL). MM: molecular weight maker.