The assembly and maintenance of eukaryotic flagella and cilia and cilia-based signaling depends on Intraflagellar Transport (IFT),a conserved bidirectional motility of protein particles composed IFT-A and IFT-B complex underneath the ciliary membrane.The function of IFT has been revealed by mutations of genes encoding various IFT polypeptides.In this study,we explore the function and mechanism of IFT54,an IFT-B subunit in ciliogenesis and IFT complex assembly in Chlamydomonas.An ift54 mutant generated by DNA insertional mutagenesis is apparently a null mutant as evidenced by lack of protein expression.ift54 cells form palmelloid colonies and do not have visible flagella.Electron microscopy reveals that the mutant doesnt form flagella beyond the transition zone.The aflagellate phenotype can be rescued by transformation of HA-tagged IFT54.IFT54 is localized in the peribasal body region and along the length of flagella similar to other IFT proteins.Sucrose gradient analysis and co-immunoprecipitation assay confirm that Chlamdyomonas IFT54 is a subunit of IFT-B.Loss of IFT54 doesnt influence the stability of the IFT complex and cellular localization of other IFT proteins.In contrast,the stability of IFT54 depends on the integrity of IFT complex B.IFT54 interacts only with IFT20 via the coil-coil domain shown by yeast two hybrid analysis.Loss of IFT54 results in undetectable level of IFT20.The coiled-coil region but not the N-terminal calponin homology domain is essential for proper intracellular localization and function of IFT54.This study provides insights of IFT54 in ciliogenesis and IFT assembly.