Substrate-induced condensation activates plant TIR domain proteins. Plant TIR domain proteins mediate recognition of pathogen effectors through their C-terminal domains. Effector binding enables TIR-encoded enzymatic activities required for immunity. Truncated TNL proteins lack effector-sensing domains but retain similar activities. The mechanism of TIR activation remains unclear. Here, it is shown that binding of NAD+ and ATP induces phase separation of TIR domain proteins in vitro. A similar condensation occurs in planta in response to pathogen inoculation. TIR condensates are mediated by conserved self-association interfaces and intrinsically disordered loops. Mutations disrupting TIR condensates impair cell death activity. Phase separation is a mechanism for TIR activation and provides insight into substrate-induced autonomous activation of TIR signalling for immunity. Plant innate immune receptors include PRRs and NLRs. PRRs detect pathogen-associated molecular patterns, while NLRs detect effectors inside plant cells. NLRs are divided into CNLs and TNLs. TNLs form resistosomes that enable TIR-encoded NADase activity. TNL resistosomes have ADP-ribosylation activity. TIRs catalyse the production of small molecules that activate EDS1-PAD4 or EDS1-SAG101 dimers, which interact with downstream NLRs to mediate immunity. Truncated TNLs, including TIR-only proteins, are present in plant genomes. These proteins can trigger NADase-dependent cell death. TIR-only proteins like RBA1 in Arabidopsis respond to pathogen effectors to trigger EDS1-dependent ETI. Self-association mediated by conserved interfaces is important for TIR activities. TIR signalling also plays a role in PTI and abiotic stress responses. PTI elicitors induce TIR signalling, which boosts PTI response. Pathogen effector binding is required to stimulate TNL enzymatic activities. Substrate-induced phase separation of TIR domain proteins was observed in vitro and in planta. NAD+ and ATP induce phase separation of TIR proteins. RPP1-TIR-GFP forms liquid-like droplets in response to NAD+ and ATP. FRAP assays show dynamic internal environments of droplets. ATP induces phase separation with higher activity than NAD+. NAD+ and ATP together also induce phase separation. Multiple interfaces mediate TIR condensation. The BB-loop is a potential intrinsically disordered region in TIR proteins. Mutations in the BB-loop disrupt phase separation. The BB-loop is conserved in TIR proteins and is important for phase separation. Mutations in the AE interface also impair NADase activity. These results suggest that multiple interfaces are involved in TIR condensation. TIR domain proteins form condensates in vivo. RPP1-TIR, RBA1, and TX14 formSubstrate-induced condensation activates plant TIR domain proteins. Plant TIR domain proteins mediate recognition of pathogen effectors through their C-terminal domains. Effector binding enables TIR-encoded enzymatic activities required for immunity. Truncated TNL proteins lack effector-sensing domains but retain similar activities. The mechanism of TIR activation remains unclear. Here, it is shown that binding of NAD+ and ATP induces phase separation of TIR domain proteins in vitro. A similar condensation occurs in planta in response to pathogen inoculation. TIR condensates are mediated by conserved self-association interfaces and intrinsically disordered loops. Mutations disrupting TIR condensates impair cell death activity. Phase separation is a mechanism for TIR activation and provides insight into substrate-induced autonomous activation of TIR signalling for immunity. Plant innate immune receptors include PRRs and NLRs. PRRs detect pathogen-associated molecular patterns, while NLRs detect effectors inside plant cells. NLRs are divided into CNLs and TNLs. TNLs form resistosomes that enable TIR-encoded NADase activity. TNL resistosomes have ADP-ribosylation activity. TIRs catalyse the production of small molecules that activate EDS1-PAD4 or EDS1-SAG101 dimers, which interact with downstream NLRs to mediate immunity. Truncated TNLs, including TIR-only proteins, are present in plant genomes. These proteins can trigger NADase-dependent cell death. TIR-only proteins like RBA1 in Arabidopsis respond to pathogen effectors to trigger EDS1-dependent ETI. Self-association mediated by conserved interfaces is important for TIR activities. TIR signalling also plays a role in PTI and abiotic stress responses. PTI elicitors induce TIR signalling, which boosts PTI response. Pathogen effector binding is required to stimulate TNL enzymatic activities. Substrate-induced phase separation of TIR domain proteins was observed in vitro and in planta. NAD+ and ATP induce phase separation of TIR proteins. RPP1-TIR-GFP forms liquid-like droplets in response to NAD+ and ATP. FRAP assays show dynamic internal environments of droplets. ATP induces phase separation with higher activity than NAD+. NAD+ and ATP together also induce phase separation. Multiple interfaces mediate TIR condensation. The BB-loop is a potential intrinsically disordered region in TIR proteins. Mutations in the BB-loop disrupt phase separation. The BB-loop is conserved in TIR proteins and is important for phase separation. Mutations in the AE interface also impair NADase activity. These results suggest that multiple interfaces are involved in TIR condensation. TIR domain proteins form condensates in vivo. RPP1-TIR, RBA1, and TX14 form