Directed Evolution of FLS2 towards Novel Flagellin Peptide Recognition
Fig 5
Five elevated-flg22-response alleles of FLS2 identify two amino acid positions that confer increased recognition of Erwinia amylovora flg22 (E22).
All bars present pooled data from two to three independent experiments. (A) Seedling growth inhibition assay of FLS2-mediated response to flg peptides. At least eight T1 plants per treatment per genotype were tested. Asterisks: significant differences of E22-treated samples from mock-treated samples within a given genotype (ANOVA, p < .05); ND: not determined. (B) Response to X22 and E22 peptides in a seedling growth inhibition assay. At least eight T1 plants per treatment per genotype were tested. None of the observed responses were significantly different from mock-treated samples of the same genotype (ANOVA, all p>.05). (C) Location of FLS2 residues relative to flg22 residues in FLS2/flg22/BAK1 co-crystal structure of [36]. Tan: FLS2 LRR main chain (cartoon representation; arrows = beta-strand regions; twelve repeats of the LRR are shown; amino-terminus to right and juxtamembrane region to left in picture); red: wild-type E321, S345 and F435 residues that were mutated in elevated-response FLS2 proteins; tan side-chains: adjacent S320 and H344 residues that bind flg22; blue: flg22 main chain and side chains in stick representation (flg22 carboxy-terminus is in upper left). Portions of BAK1, FLS2 and an SO4 from co-crystal structure would occupy foreground in upper left and are removed from view. (D) ROS burst in response to peptides. Area under curve for 30 min. ROS burst for the designated allele and treatment divided by mean area for mock-treated leaf discs of same genotype in same experiment. Assays performed on different days are shown in separate graphs. Asterisks: significantly more total ROS production relative to mock-treated controls within a given genotype (ANOVA, p < .05). (E) Bacterial growth assays using in six-week old T2 plants containing receptors of interest (as well as Col-0 and fls2- controls). Rosette leaves were infiltrated with the indicated peptides 24 hours before infiltration with 105 cfu/mL P. syringae pv. tomato DC3000. Two days later, leaf samples were removed and bacterial titers were determined.