A RALF22-like Peptide Coordinates Salt Tolerance and Disease Susceptibility in Poplar (Populus davidiana × P. bolleana ‘Shanxin’)

Rapid alkalinization factor (RALF) peptides are recognized as multifunctional regulators of plant stress responses, yet their roles in woody species remain poorly defined. Here, we identified a RALF22-like peptide from poplar ‘Shanxin’ (Populus davidiana × P. bolleana; PdbRALF22-like) and investigated its roles in salt tolerance and disease resistance. Synthetic PdbRALF22-like peptide elicited a rapid ROS burst in poplar leaf discs. In Nicotiana benthamiana, which was otherwise unresponsive to the peptide, transient expression of either of two poplar FERONIA-like receptor kinases (PdbFER-like-1 and PdbFER-like-2) enabled peptide-triggered ROS production, consistent with receptor-matched responsiveness in a heterologous context. Using CRISPR/Cas9, we generated a PdbRALF22-like knockout line and assessed salt tolerance in vitro and soil-grown assays. Under salinity, the mutant showed sustained rooting at high NaCl concentrations and improved growth relative to wild type. After 0.2 M NaCl treatment, soil-grown mutant plants exhibited reduced wilting and leaf injury. Evans Blue, DAB, and NBT staining indicated reduced membrane damage and lower accumulation of hydrogen peroxide and superoxide in the mutant. Significantly, the same knockout line displayed increased susceptibility to infection by the poplar leaf spot fungus, with larger lesions and higher pathogen biomass, accompanied by reduced ROS output and lower induction of the defense marker gene PdbPR1. Collectively, PdbRALF22-like negatively regulates salt tolerance while contributing positively to disease resistance, and represents a regulatory node linking salinity tolerance and disease susceptibility in poplar ‘Shanxin’, with poplar FER-like receptors providing a plausible route for peptide-triggered ROS signaling. This work expands our understanding of RALF peptide signaling in woody plants.