Cis-Regulatory Editing of Peptide Signaling Rewires Plant Growth-Defense Balance

Precise cis-regulatory control of gene expression is essential for balancing plant growth and stress responses. In Arabidopsis thaliana, PLANT PEPTIDE CONTAINING SULFATED TYROSINE (PSY) peptides and their receptors (PSYRs) mediate growth–stress trade-offs, yet the transcriptional regulation of these genes remains poorly understood. Here, we mapped transcription factor (TF)–promoter interactions for nine PSY and three PSYR genes by combining high-throughput yeast one-hybrid screening with DAP-seq data, uncovering 1,207 interactions that reveal both shared and gene-specific regulatory relationships. Functional analysis of 25 TF mutants identified 12 regulators that significantly influence shoot and root growth, most acting as repressors. Of these, CYTOKININ RESPONSE FACTOR 10 (CRF10) emerged as a strong growth inhibitor. We found a CRF10 binding motif in the PSYR3 promoter using DAP-seq data and validated by eY1H. Guided by these insights, we applied CRISPR/Cas9-mediated promoter editing to precisely delete functional TF-binding sites. Removal of this motif, or its surrounding region, reduced PSYR3 expression and enhanced root growth, yielding variants that retained root length comparable to the crf10 mutant but displayed reduced susceptibility to Pseudomonas syringae pv. tomato (Pst) DC3000. Together, our results define the global TF–promoter interaction network of the PSYR/PSY pathway and identify CRF10 as a key transcriptional regulator of PSYR3-mediated signaling. More broadly, our work demonstrates that cis-regulatory editing can systematically fine-tune gene expression, generate predictable growth traits, and provide a TFBS-targeted framework for engineering plant growth–defense balance.