Pollen tubes are crucial for angiosperm plants, as they deliver sperm gametes for the essential process of double fertilization. Understanding the molecular mechanisms behind pollen tube germination and growth is critical
however, these processes remain partially elucidated in monocot cereal crops. Rapid Alkalinization Factor (RALF), a small peptide of about 5 kDa, binds to the CrRLK1L receptor and plays a role in various plant physiological processes, including reproduction and tip growth. Recently, we reported that OsRALF17 and OsRALF19 binds to the OsMTD2, pollen specific CrRLK1L member, and regulates pollen tube growth. In this study, we demonstrate that Ruptured Pollen tube (RUPO), another CrRLK1L member, is also a putative receptor for OsRALF17 and OsRALF19, and propose the formation of a receptor complex with OsMTD2. In tobacco epidermal cells, OsMTD2 and RUPO were co-localized at both the plasma membrane (PM) and the nuclear membrane. Additionally, we generated a RUPO-tagged line driven by its native promoter to visualize subcellular localization during pollen tube growth. RUPO localizes a tip-enriched distribution, with intense fluorescence at the tip's PM and cytoplasm in pollen tube. Upon treatment with synthetic OsRALF17M and OsRALF19M peptides, a reduction in the signal near the PM was observed, suggesting a potential response to these peptides. Our data support the role of RUPO as a candidate receptor for OsRALF17 and OsRALF19 in rice pollen tubes, thereby suggesting a novel mechanism for these RALFs in regulating pollen tube function. Additionally, we observed a significant delay in pollen tube burst time upon treatment with synthetic OsRALF17M and synthetic OsRALF19M. We propose that investigating this phenomenon may provide further insights into the specific signaling pathways mediated by these RALFs.