Fig. 2: Engineering expanded ligand perception from QvFLS2 recognizing Ralstonia and Dickeya pathogens.
From: Unlocking expanded flagellin perception through rational receptor engineering
a, ROS production after treatment with flg22 variants. FLS2s were transiently expressed in the N. benthamiana fls2-1/2 CRISPR–Cas9 mutant. Each data point represents an average maximum RLU from four plants with four leaf disks per plant. Normalized ROS levels were calculated by adjusting maximum RLU averages to a 0 to 100,000 scale, referencing controls (water = 0 and Pae flg22 = 100,000) for each receptor. One-way analysis of variance and Dunnett’s multiple comparison were performed using non-scaled data, P values are marked for each column. Error bars denote s.e.m. b, AlphaFold3 model of the LRR extracellular domains of QvFLS2 and co-receptor NbSERK3A with Ralstonia solanacearum (Rso_1) flg22 peptide. The colour represents predicted local distance difference test (pLDDT). c, RCM of 42 FLS2 homologues in the Fagales order, showing conservation levels from high (yellow) to low (dark blue). White asterisks mark the residues transferred from QvFLS2 to FcFLS2 in synthetic FcFLS2 (SynFcFLS213Qv), which are associated with the expanded ligand detection. Colour bars indicate interacting regions of the flg22 ligand or co-receptor SERK3. d, Representations of FLS2 variants. Colour bars indicate residue transfers. The numbers of residues transferred are superscripted. e, ROS production after treatment with flg22 variants (100 nM). FLS2s were transiently expressed in the N. benthamiana fls2-1/2 CRISPR–Cas9 mutant. Normalized ROS levels were calculated by adjusting RLU averages to a 0 (water) to 100 (Pae flg22) scale. Data points below a value of 10 are depicted as white bubbles. f, AlphaFold3 model of SynFcFLS213Qv LRR and co-receptor NbSERK3A LRR with Rso_1 flg22 peptide. The N terminus and C terminus of flg22 are labelled. g, ROS production after treatment of flg22 variants with a concentration gradient as described in a. h, Phosphorylation of MAPK induced by flg22 variants (100 nM) for different receptors. All experiments were repeated independently at least three times with similar results. CBB staining indicates protein loading. C′, flg22 C terminus; CT, C-terminal LRR domain; Fc, Fagus crenata; N′, flg22 N terminus; NT, N-terminal LRR domain.
