PMC

Genotyping of BAK1 (Y610F)-Flag lines in bak1-4 bak1-4/bkk1-1 bkk1-1 background. (A,B) Genomic organization of the T-DNA insertion knockout lines, bak1-4 (SALK_116202) and bkk1-1 (SALK_057955), both in the Col-0 ecotype. The position of each T-DNA insertion is depicted by an inverted triangle, and arrows showing positions of primers used for genotyping. (C) PCR using BAK1 gene specific primers (upper gel panel) and a T-DNA specific primer (lower gel panel). (D) PCR using BKK1 gene specific primers (upper gel panel) and a T-DNA specific primer (lower gel panel). The three lines used for genotyping are independent lines; WT is wild-type Col-0 that served as a positive control for gene specific primers and negative control for T-DNA specific primers.

Quantitative real time PCR analysis of genes potentially down regulated in BAK1 (Y610F)-Flag plants relative to BAK1-Flag plants. Transcript accumulation of (A) LCR67, (B) PCC1, (C) BT5, and (D) AT5g39180 in BAK1-Flag and three independent line of BAK1 (Y610F)-Flag. Quantitative real-time polymerase chain reaction was used to determine the abundance of the different transcripts relative to the abundance of the ACTIN2 transcript. Each data point represents mean ± standard deviation (SD) of n = 4. Statistical significance of differences with respect to BAK1-Flag were assessed by Welch test with probability values of ^∗∗∗∗p < 0.0001, ^∗∗p < 0.001, ^∗p < 0.01 and ns, not significant indicated above the respective bars. Experiments were done two times with similar results.

Similar growth and BL responses in transgenic plants expressing BAK1-Flag or BAK1 (Y610F)-Flag in the double null background. (A) Phenotype of 18- and 45-day-old Col-0, BAK1-Flag and BAK1 (Y610F)-Flag plants (in the bak1-4 bak1-4/bkk1-1 bkk1-1 double null background) grown in a normal long day photoperiod. (B) Immunoblot analysis indicates similar expression levels of the BAK1-Flag transgene in the different genotypes. Seedlings were grown in liquid MS medium for 10 days. Protein was immunoprecipitated using immobilized anti-Flag antibodies of solubilized microsomal membranes, and immunoblotting was performed with anti-Flag antibodies. The reactive band at 50 kDa (asterisk) is the heavy chain of the immobilized M2 antibody released from the Anti-Flag M2 Beads by elution with SDS-PAGE sample buffer containing reductant. (C) Dose-dependent hypocotyl elongation of 7 days old seedlings of BAK1-Flag, BAK1 (Y610F)-Flag and det2 at 0, 10, 100, and 1000 nM BL. Seedlings were grown in the long day photoperiod.

BAK1 (Y610F)-Flag plants respond similarly to wild-type and BAK1-Flag plants in FLS2-mediated inhibition of growth and bacterial growth following inoculation. (A) Relative growth inhibition in response to 10 nM flg22 in wild-type, BAK1-Flag and BAK1 (Y610F)-Flag. Each data point represents mean ± standard deviation (SD) of n = 7, Weltch t-test (p < 0.05) was performed, ns, not significant. (B) Bacterial growth measurement at 0 and 3 days post inoculation (dpi) of hrpA^- mutant of Pst DC3000 in BAK1-Flag, BAK1 (Y610F)-Flag #1, #2, and #3 leaves. Bacterial suspension of 10⁴ cfu mL^-1 was infiltrated into the abaxial leaf surface with a needleless syringe. Bacterial numbers were counted at 3 days post inoculation. Each bar represents mean ± standard deviation (SD) of n = 12 for BAK1-Flag, BAK1 (Y610F)-Flag #1, BAK1 (Y610F)-Flag #3 and n = 8 for BAK1 (Y610F)-Flag #2. Each sample consisted of four leaf disks of 5 mm diameter taken from inoculated leaves. Experiments were done three times with similar results.

Stable expression of untagged BAK1 or BAK1 (Y610F) rescues the reduced leaf growth phenotype of the bak1-4 mutant but does not affect EFR- and FLS2-dependent PTI signaling. (A) The bak1-4/BAK1 (Y610F) plants have a wild-type-like morphology under short-day conditions. Picture of representative individuals of 35-day-old Col-0, bak1-4, bak1-4/BAK1, bak1-4/BAK1 (Y610F)#4-3, and bak1-4/BAK1 (Y610F)#7-3 plants grown under short-day conditions. Scale bar represents 2 cm. (B) The BAK1 (Y610F) lines display wild-type levels of PAMP-induced ROS in stable Arabidopsis bak1-4 mutant lines. Total ROS production in leaf disks of the indicated genotypes expressed as total relative light units (RLUs) after treatment with 100 nM elf18 (left panel) or 100 nM flg22 (right panel) for 60 min. Values are means ± SE (n = 12). Statistical analysis was performed using analysis of variance (ANOVA) and Bonferroni post-test (^∗∗∗p < 0.001, ^∗∗p < 0.01, ^∗p < 0.05). (C) The BAK1 (Y610F) lines display wild-type levels elf18- (left panel) and flg22-induced (right panel) MAPK activation. Phosphorylation of MAPKs at 5 min after PAMP treatment, as shown by Western Blot using an anti-p44/42-ERK antibody. Individual MPKs are identified by molecular weight and indicated by arrows. The membranes were blotted with anti-BAK1 antibodies and subsequently stained with Coomassie colloidal blue for loading control. (D) BAK1 (Y610F) lines display wild-levels of elf18 (left panel) and flg22 (right panel)-induced seedling growth inhibition. Growth is represented as relative fresh weight to the untreated control. Results are means + SE (n = 8).