The insect effector CSPs activate an atypical plant TNL immune receptor.

key messageCSP-mediated activation of RCSP involves noncanonical structural and catalytic mechanisms-including BB-loop deletion, α5α6-loop insertion, and atypical catalytic residues within the RCSP-TIR domain. Chemosensory proteins (CSPs), a conserved effector family in phloem-feeding insects such as aphids and the brown planthopper (Nilaparvata lugens), induce dwarfism in Nicotiana benthamiana through recognition by the Toll-interleukin-1 receptor (TIR) domain-containing nucleotide-binding leucine-rich repeat receptor (TNL) RCSP. However, CSP-mediated RCSP activation and enzymatic mechanism remain unknown. Here, we show that TIR domain of RCSP (RCSP-TIR) exhibits unique features, including a 9-amino acid deletion in the loop between β-strand B and α-helix B (BB-loop), an 8-amino acid insertion in the loop between α5 and α6 (α5α6-loop), and substitution of the canonical catalytic glutamate with glutamine (E87Q). Structural analysis demonstrates that BB-loop deletion disrupts the canonical NAD⁺-binding pocket and prevents standard TIR tetramerization. Strikingly, AlphaFold3/DMFold modeling reveals that RCSP compensates via α5α6-loop insertion, forming a novel interface that stabilizes an atypical tetrameric architecture. Integrated approaches combining molecular docking and site-directed mutagenesis demonstrate that RCSP may utilize a noncanonical NAD⁺-binding pocket and depend on dual catalytic residues (D86/Q87) to mediate weak cell death. Evolutionary analysis classifies RCSP-TIR homologs into four distinct clades, highlighting Solanaceae-specific adaptations. These findings elucidate a novel plant-insect interaction mechanism in which RCSP's structural diversification enables CSPs recognition and immune signaling reprogramming.