Redefinition of the toll-like receptor repertoire in Ciona robusta through genomic, structural, and expression analyses

Background Toll-like receptors (TLRs) are essential components of innate immunity, mediating the recognition of pathogen-associated molecular patterns (PAMPs) through extracellular leucine-rich repeat (LRR) domains and initiating signaling via intracellular Toll/interleukin-1 receptor (TIR) domains. In the ascidian Ciona robusta , two canonical TLRs (CiTLR1 and CiTLR2) and several putative TLR-like genes (TLR3, -4, -6, -7, -13) have been annotated; however, their authenticity has remained uncertain due to limited structural and functional validation. Methods we systematically reanalyzed the Ciona genome using the latest nearly complete assembly (HT genome) in combination with domain prediction, three-dimensional structural modeling, and transcriptomic expression profiling. Results Genomic mapping and sequence comparison demonstrated that TLR13 is identical to CiTLR1, while TLR3, -6, and -7 lack a complete TIR domain, indicating that these are not canonical TLRs. We further identified a novel TLR gene, CiTLRs1, located approximately 42 kb from CiTLR1 on chromosome 14, which encodes all essential structural features including LRR and TIR domains. AlphaFold3 structural predictions confirmed that CiTLR1, CiTLR2, and CiTLRs1 possess canonical solenoid LRR folds and typical TIR domain architectures. In addition, we found no convincing evidence that CiTLR3, CiTLR6, or CiTLR7 function as soluble TLRs. Transcriptomic analyses revealed distinct tissue-specific expression profiles of these genes, suggesting nonredundant immune functions. Conclusions Our findings revise the repertoire of bona fide TLRs in Ciona to three (CiTLR1, CiTLR2, CiTLRs1) and emphasize the risk of overestimating TLR diversity based solely on sequence homology without domain and functional validation. This work refines the structural and functional landscape of ascidian TLRs.