The study investigates the mechanism by which substrate-induced condensation activates plant TIR domain proteins. The authors demonstrate that binding of substrates NAD^+^ and ATP induces phase separation of TIR domain proteins both in vitro and in planta. This condensation is mediated by conserved self-association interfaces and a predicted intrinsically disordered loop region (IDR) in the TIR domain. Mutations disrupting this IDR impair the cell death activity of TIR domain proteins. The findings reveal phase separation as a mechanism for the activation of TIR domain proteins and provide insights into substrate-induced autonomous activation of TIR signaling to confer plant immunity. The study also highlights the importance of multi-valency and IDRs in phase separation, suggesting that these mechanisms may be evolutionarily conserved for TIR proteins to modulate their enzymatic activities.The study investigates the mechanism by which substrate-induced condensation activates plant TIR domain proteins. The authors demonstrate that binding of substrates NAD^+^ and ATP induces phase separation of TIR domain proteins both in vitro and in planta. This condensation is mediated by conserved self-association interfaces and a predicted intrinsically disordered loop region (IDR) in the TIR domain. Mutations disrupting this IDR impair the cell death activity of TIR domain proteins. The findings reveal phase separation as a mechanism for the activation of TIR domain proteins and provide insights into substrate-induced autonomous activation of TIR signaling to confer plant immunity. The study also highlights the importance of multi-valency and IDRs in phase separation, suggesting that these mechanisms may be evolutionarily conserved for TIR proteins to modulate their enzymatic activities.