Journal: bioRxiv

Article Title: Anti-Malaria Antibody Engineering Broadens Recognition Motifs and Reveals New Homotypic Interactions that Enhance Protective Breadth

doi: 10.1101/2025.09.30.679367

Figure Lengend Snippet: (A) The L9 antibody was used as template for multiple rounds of directed evolution for affinity improvement against malaria antigens. First, site-saturation mutagenesis (SSM) and sorting was used against 0.5 nM Pep22 (2 NVDPs). Next, enriched variants further diversified using SSM and DNA shuffling, followed by sorting against 1.25 nM Pep22. Finally, the enriched libraries were shuffled again and sorted under increasingly stringent conditions by progressively lowering Pep22 concentrations during FACS, with a final enrichment step against NTDS_5/3 (a truncated CSP with 5 NANP / 3 NVDP repeats). (B) Representative flow cytometry plots across the screening experiments. Each population was labeled with 62.5 pM Pep22. A high-affinity gate is drawn for comparative purposes, and the percentage of the high affinity population is shown in the upper-right quadrant. The mean fluorescence intensity ratio (MFI) is calculated as (Geomean PE / Geomean FITC ) to quantitatively represent the normalized affinity of Fab molecules. See also – .

Article Snippet: Libraries from the first round of DNA shuffle mutagenesis were labeled with 1.25 nM biotinylated Peptide 22 (NANPNVDPNANPNVD) conjugated with streptavidin-PE (S21388, Invitrogen), and sorted for three rounds by FACS, as described above.

Techniques: Mutagenesis, Flow Cytometry, Labeling, Fluorescence

Journal: Nature Plants

Article Title: Reverse engineering of the pattern recognition receptor FLS2 reveals key design principles of broader recognition spectra against evading flg22 epitopes

doi: 10.1038/s41477-025-02050-5

Figure Lengend Snippet: a , Sequences of flg22 Atum and flg22 Pa , and ROS production (in RLU) induced by VvFLS2 and VrFLS2XL in response to these peptides ( n = 12 leaf discs). Mutations in flg22 Atum are highlighted in red, with reported key mutations (15N, 18Y, 19W, 20S) in boldface and shaded. b , Total ROS production triggered by VvFLS2 in response to flg22 Pa and increasing concentrations of flg22 Atum ( n = 8 leaf discs). c , AlphaFold3-predicted structure of VrFLS2XL ECD -flg22 Atum -NbBAK1 ECD and the segmentation scheme of VrFLS2XL for domain swapping (ECD, extracellular domain; EJM, EJM domain). NbBAK1 is also known as NbSERK3B. The ipTM and pTM values stand for the interface predicted template modelling (ipTM) score and the predicted template modelling (pTM) score. d , Flg22 Atum -induced ROS production of VvFLS2-VrFLS2XL chimeras ( n = 36, 52, 16, 28, 16, 40, 76, 48, 36, 64 leaf discs, from top to bottom). e , Flg22 Atum -induced ROS production of chimera 0100 ( n = 28 leaf discs). f , Phosphorylation of MAPKs by FLS2 variants in response to flg22 Pa and flg22 Atum . g , Histochemical (left) and fluorometric (right) GUS assays showing the effects of FLS2 variants on restricting the T-DNA transfer of A. tumefaciens carrying a GUS reporter gene ( n = 7 biological replicates for the fluorometric assay). White dashed lines indicate regions where FLS2s are expressed. Dark blue colouration indicates high GUS activity and a higher degree of T-DNA transfer. h , Significant restriction of A. tumefaciens growth by VrFLS2XL and 0100 compared to VvFLS2 measured by counting of colony-forming units (CFU) ( n = 5 biological replicates). For bar plots and line charts, data are shown as mean ± s.e.m., except for h , where mean ± s.d. is used. For box plots: centre line, median; box limits, upper and lower quartiles; whiskers, 1.5 times interquartile range; points, outliers. Kruskal–Wallis test followed by Dunn’s multiple comparisons test was used for statistical analysis in b , d , g and h . See Source data for exact P values. Significant differences at P < 0.05 are denoted by letters. Two-tailed Mann–Whitney test was used for statistical analysis in e . All experiments were repeated three times with similar results, except for h , which was repeated twice.

Article Snippet: LRR12-19 VvFLS2 fragments carrying computationally predicted polymorphic residue sets (V1, V2, V3), the LRR20-EJM GmFLS2a fragment carrying YHR and YHAIR (F579Y, Q600H, S603A, M626I, H723R) mutations and the codon-optimized LRR20-EJM GmFLS2b fragment for DNA shuffling were synthesized by Twist Bioscience.

Techniques: Phospho-proteomics, Activity Assay, Two Tailed Test, MANN-WHITNEY

Journal: Nature Plants

Article Title: Reverse engineering of the pattern recognition receptor FLS2 reveals key design principles of broader recognition spectra against evading flg22 epitopes

doi: 10.1038/s41477-025-02050-5

Figure Lengend Snippet: a , Schematic diagram of the DNA shuffling workflow. See Methods section for detailed explanations. For illustrative purposes only, the numbers on the leaves indicate the serial numbers of the shuffled variants tested, while the dashed lines mark the infiltrated regions. b , Summary of the sequences and performances of 30 shuffled variants. c , Analysis of DNA shuffling results. See the Methods section for detailed explanations of this plot. d , Flg22 Rso -induced ROS production of GmFLS2a with single and double mutations of H600Q and S603A ( n = 12 leaf discs). e , Effects of A603S and H600Q on the flg22 Rso -induced ROS response of chimera aaba ( n = 12 leaf discs). f , Effects of DNA shuffling-predicted enhancer and ‘suppressor’ polymorphisms on flg22 Rso -induced ROS production ( n = 36, 80, 36, 44, 36 leaf discs, from top to bottom). See c for the definitions of E, P1, P2, P3 and N. g , Comparison of total flg22 Rso -induced ROS production between GmFLS2b and SuperGmFLS2 ( n = 138 and 130 leaf discs, from left to right). SuperGmFLS2 combines all the GmFLS2b-originated (orange) regions/polymorphic residues with positive effects on flg22 Rso recognition. h , Co-IP of NbBAK1 with wild-type GmFLS2a and two engineered variants. For bar plots and line charts, data are presented as mean. For box plots: centre line, median; box limits, upper and lower quartiles; whiskers, 1.5 times interquartile range; points, outliers; ‘+’, mean. Kruskal–Wallis test followed by Dunn’s multiple comparisons test was used for statistical analysis in d – f with significant differences at P < 0.05 indicated by letters. See Source data for exact P values. Two-tailed Mann–Whitney test was used for g . ROS experiments in d – g were repeated three times with similar results. Co-IP experiment in h was repeated twice with similar results. Panel a created with BioRender.com .

Article Snippet: LRR12-19 VvFLS2 fragments carrying computationally predicted polymorphic residue sets (V1, V2, V3), the LRR20-EJM GmFLS2a fragment carrying YHR and YHAIR (F579Y, Q600H, S603A, M626I, H723R) mutations and the codon-optimized LRR20-EJM GmFLS2b fragment for DNA shuffling were synthesized by Twist Bioscience.

Techniques: Comparison, Co-Immunoprecipitation Assay, Two Tailed Test, MANN-WHITNEY