Single‐Cell Transcriptomics Reveals FLS2‐Dependent Hypoxia Signaling and ERF13‐Mediated Transcription During flg22‐Triggered Immunity

The flagellin peptide flg22 activates FLAGELLIN-SENSING 2 (FLS2)-mediated immunity in Arabidopsis, leading to growth inhibition and oxidative burst. While these responses are well-studied, their cell-type-specific regulation remains poorly understood. Using single-cell RNA sequencing, genetics, and phenotyping, we systematically mapped flg22-induced responses. flg22 suppressed growth and elevated reactive oxygen species (ROS) in wild-type, but not in fls2 mutants. Epidermal (EP_3) and mesophyll (MPC_2) cells showed FLS2-dependent transcriptional reprogramming. Pseudotime analysis revealed developmental trajectories toward immune-activated states. flg22 also induced a hypoxia-like response; hypoxic signaling mutants (ate1, prt6, zpr2) showed reduced flg22 sensitivity, indicating crosstalk between immune and hypoxia pathways. ERF13 was identified as a central regulator: erf13 mutants impaired flg22-triggered ROS and growth inhibition but enhanced effector-triggered immunity (ETI), while overexpressors showed stronger pattern-triggered immunity (PTI). flg22 altered ploidy and cell-cycle gene expression in WT, which was stabilized in ate1 and erf13 mutants. Cell-cycle mutants sim smr and e2fabc enhanced flg22 responses, whereas cpr5 was less sensitive. Thus, immune, hypoxia, and ROS signals converge via ERF13 to balance immunity and growth, providing a single-cell view of spatial immune organization and stress adaptation.