Comparative analysis of rapid alkalinization factor peptide-triggered plant immunity in citrus and closely related...

Rapid alkalinization factors (RALFs) are plant-derived, cysteine-rich small peptides that play crucial roles in plant development and immunity. However, their function in citrus immunity remains unexplored. This study systematically identified RALF family members in sweet orange, a cultivated citrus species, and five closely related species known for their strong resistance to citrus diseases. Most species contained 13 RALF genes, and their corresponding RALF orthologs exhibited minimal variation in gene structure, sequence identity, conserved domains, and motifs. However, differences in cis-acting elements within promoter regions led to differences in their expression patterns under biotic stress. Compared with sweet orange, citrus-related species exhibited a stronger and more widespread induction of RALF genes. Additionally, RALF peptides, particularly RALF11, triggered more robust immune responses in these species, including ROS bursts, mitogen-activated protein kinase activation, and the upregulation of defense-related genes. Furthermore, the basal expression levels of CrRLK1Ls, the RALF receptor homologs, were higher in citrus-related species than in sweet orange. Amplifying the RALF-CrRLK1L pathway through CrRLK1L overexpression and RALF peptide application significantly enhanced sweet orange resistance to citrus canker. These findings suggest that citrus-related species have endured selective pressure, leading to greater promoter sequence diversity, which is conducive to fine-tune gene expression in response to environmental challenges.