biostudies-literatureZhang SUniversität Zürich (University of Zurich)Universität ZürichZurich-Basel Plant Science Center1642-1657https://www.ebi.ac.uk/biostudies/studies/S-EPMC12364711biostudies-literatureUnknown11(8)In the ongoing plant-pathogen arms race, plants use pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs), while in successful pathogens, PAMPs can evolve to evade detection. Engineering PRRs to recognize evading PAMPs could potentially generate broad-spectrum and durable disease resistance. Here we reverse-engineered two natural variants of the PRR FLAGELLIN SENSING 2 (FLS2), VrFLS2XL and GmFLS2b, with extended recognition specificities towards evading flg22 variants. We identified minimal gain-of-function residues enabling blind FLS2s to recognize otherwise evading flg22 variants. We uncovered two strategies: (1) optimizing FLS2-flg22 interaction around flg22's key evasion sites and (2) strengthening direct FLS2-BAK1 interaction to overcome weak agonistic and antagonistic flg22s, respectively. In addition, we leveraged polymorphisms that enhance recognition through unknown mechanisms to engineer a superior recognition capability. These findings offer basic design principles to engineer PRRs with broader recognition spectra, paving the way for PRR engineering to generate precisely gene-edited disease-resistant crops.Nature plantsReverse engineering of the pattern recognition receptor FLS2 reveals key design principles of broader recognition spectra against evading flg22 epitopes.PMC12364711n/aKim GBender KWLiu SCaflisch AZhang SZipfel CLai HFfalseReverse engineering of the pattern recognition receptor FLS2 reveals key design principles of broader recognition spectra against evading flg22 epitopes.In the ongoing plant-pathogen arms race, plants use pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs), while in successful pathogens, PAMPs can evolve to evade detection. Engineering PRRs to recognize evading PAMPs could potentially generate broad-spectrum and durable disease resistance. Here we reverse-engineered two natural variants of the PRR FLAGELLIN SENSING 2 (FLS2), VrFLS2XL and GmFLS2b, with extended recognition specificities towards evading flg22 variants. We identified minimal gain-of-function residues enabling blind FLS2s to recognize otherwise evading flg22 variants. We uncovered two strategies: (1) optimizing FLS2-flg22 interaction around flg22's key evasion sites and (2) strengthening direct FLS2-BAK1 interaction to overcome weak agonistic and antagonistic flg22s, respectively. In addition, we leveraged polymorphisms that enhance recognition through unknown mechanisms to engineer a superior recognition capability. These findings offer basic design principles to engineer PRRs with broader recognition spectra, paving the way for PRR engineering to generate precisely gene-edited disease-resistant crops.2025-01-01T00:00:00Z2025 Aug2026-05-02T03:46:56.255Z2026-04-07T17:36:06.709ZS-EPMC123647114072166810.1038/s41477-025-02050-5