RESUMO
Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors with an N-terminal Toll/interleukin-1 receptor (TIR) domain mediate recognition of strain-specific pathogen effectors, typically via their C-terminal ligand-sensing domains1. Effector binding enables TIR-encoded enzymatic activities that are required for TIR-NLR (TNL)-mediated immunity2,3. Many truncated TNL proteins lack effector-sensing domains but retain similar enzymatic and immune activities4,5. The mechanism underlying the activation of these TIR domain proteins remain unclear. Here we show that binding of the TIR substrates NAD+ and ATP induces phase separation of TIR domain proteins in vitro. A similar condensation occurs with a TIR domain protein expressed via its native promoter in response to pathogen inoculation in planta. The formation of TIR condensates is mediated by conserved self-association interfaces and a predicted intrinsically disordered loop region of TIRs. Mutations that disrupt TIR condensates impair the cell death activity of TIR domain proteins. Our data reveal phase separation as a mechanism for the activation of TIR domain proteins and provide insight into substrate-induced autonomous activation of TIR signalling to confer plant immunity.
Assuntos
Trifosfato de Adenosina , Arabidopsis , NAD , Nicotiana , Separação de Fases , Proteínas de Plantas , Domínios Proteicos , Trifosfato de Adenosina/metabolismo , Arabidopsis/genética , Arabidopsis/imunologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/imunologia , Proteínas de Arabidopsis/metabolismo , Morte Celular , Mutação , NAD/metabolismo , Nicotiana/genética , Nicotiana/imunologia , Nicotiana/metabolismo , Proteínas NLR/química , Proteínas NLR/genética , Proteínas NLR/imunologia , Proteínas NLR/metabolismo , Doenças das Plantas/imunologia , Imunidade Vegetal/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas , Domínios Proteicos/genética , Receptores Imunológicos/química , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Transdução de Sinais , Receptores Toll-Like/química , Receptores de Interleucina-1/químicaRESUMO
Nucleotide-binding leucine-rich repeat (NLR) proteins play a pivotal role in plant immunity by recognizing pathogen effectors1,2. Maintaining a balanced immune response is crucial, as excessive NLR expression can lead to unintended autoimmunity3,4. Unlike most NLRs, the plant NLR required for cell death 2 (NRC2) belongs to a small NLR group characterized by constitutively high expression without self-activation5. The mechanisms underlying NRC2 autoinhibition and activation are not yet understood. Here we show that Solanum lycopersicum (tomato) NRC2 (SlNRC2) forms dimers and tetramers and higher-order oligomers at elevated concentrations. Cryo-electron microscopy shows an inactive conformation of SlNRC2 in these oligomers. Dimerization and oligomerization not only stabilize the inactive state but also sequester SlNRC2 from assembling into an active form. Mutations at the dimeric or interdimeric interfaces enhance pathogen-induced cell death and immunity in Nicotiana benthamiana. The cryo-electron microscopy structures unexpectedly show inositol hexakisphosphate (IP6) or pentakisphosphate (IP5) bound to the inner surface of the C-terminal leucine-rich repeat domain of SlNRC2, as confirmed by mass spectrometry. Mutations at the inositol phosphate-binding site impair inositol phosphate binding of SlNRC2 and pathogen-induced SlNRC2-mediated cell death in N. benthamiana. Our study indicates a negative regulatory mechanism of NLR activation and suggests inositol phosphates as cofactors of NRCs.
Assuntos
Microscopia Crioeletrônica , Modelos Moleculares , Proteínas NLR , Nicotiana , Imunidade Vegetal , Proteínas de Plantas , Multimerização Proteica , Solanum lycopersicum , Nicotiana/metabolismo , Nicotiana/imunologia , Proteínas NLR/metabolismo , Proteínas NLR/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Solanum lycopersicum/metabolismo , Solanum lycopersicum/imunologia , Ácido Fítico/metabolismo , Ácido Fítico/química , Morte Celular , Ligação Proteica , MutaçãoRESUMO
Polygalacturonase-inhibiting proteins (PGIPs) interact with pathogen-derived polygalacturonases to inhibit their virulence-associated plant cell wall-degrading activity but stimulate immunity-inducing oligogalacturonide production. Here we show that interaction between Phaseolus vulgaris PGIP2 (PvPGIP2) and Fusarium phyllophilum polygalacturonase (FpPG) enhances substrate binding, resulting in inhibition of the enzyme activity of FpPG. This interaction promotes FpPG-catalyzed production of long-chain immunoactive oligogalacturonides, while diminishing immunosuppressive short oligogalacturonides. PvPGIP2 binding creates a substrate binding site on PvPGIP2-FpPG, forming a new polygalacturonase with boosted substrate binding activity and altered substrate preference. Structure-based engineering converts a putative PGIP that initially lacks FpPG-binding activity into an effective FpPG-interacting protein. These findings unveil a mechanism for plants to transform pathogen virulence activity into a defense trigger and provide proof of principle for engineering PGIPs with broader specificity.