Genome-wide identification and characterization of Rapid alkalinization factors (RALFs) in tomato and function analysis of SlRALF2/3 in immunity

The RAPID ALKALINIZATION FACTORs (RALFs) are a crucial group of peptides in plants, regulating growth, development, and stress responses. However, the functions of RALFs in tomato have not been comprehensively explored. In this study, a total of 12 SlRALF genes were identified. Phylogenetic analysis revealed that RALF members from six dicots clustered into eight subfamilies. The gene structures and conserved motifs of SlRALFs were further examined. Expression analysis of SlRALFs in different tissues and fruit development showed that eight SlRALFs had higher expression levels in flower. Additionally, the expression of SlRALFs was induced by various biotic and abiotic stresses and plant hormones, suggesting that SlRALFs were involved in diverse physiological functions and hormone signaling pathways. Subcellular localization assay revealed that SlRALFs were secreted into apoplast. The interactions between SlRALFs and the receptor FERONIA (FER) were analyzed. Transient expression of SlRALF2 and SlRALF3 in Nicotiana benthamiana leaves induced reactive oxygen species (ROS) levels and defense-related genes expression. Furthermore, both SlRALF2 and SlRALF3 enhanced tobacco resistance to Botrytis cinerea , indicating their important roles in plant immunity. This study provides new insights into the potential biological roles of SlRALFs in tomato, expanding our understanding on their function in plants.