RESUMEN
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.
Asunto(s)
Adenosina Trifosfato , Arabidopsis , NAD , Nicotiana , Separación de Fases , Proteínas de Plantas , Dominios Proteicos , Adenosina Trifosfato/metabolismo , Arabidopsis/genética , Arabidopsis/inmunología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/inmunología , Proteínas de Arabidopsis/metabolismo , Muerte Celular , Mutación , NAD/metabolismo , Nicotiana/genética , Nicotiana/inmunología , Nicotiana/metabolismo , Proteínas NLR/química , Proteínas NLR/genética , Proteínas NLR/inmunología , Proteínas NLR/metabolismo , Enfermedades de las Plantas/inmunología , Inmunidad de la Planta/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/inmunología , Proteínas de Plantas/metabolismo , Regiones Promotoras Genéticas , Dominios Proteicos/genética , Receptores Inmunológicos/química , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/metabolismo , Transducción de Señal , Receptores Toll-Like/química , Receptores de Interleucina-1/químicaRESUMEN
Plant pathogen-activated immune signaling by nucleotide-binding leucine-rich repeat (NLR) receptors with an N-terminal Toll/interleukin-1 receptor (TIR) domain converges on Enhanced Disease Susceptibility 1 (EDS1) and its direct partners, Phytoalexin Deficient 4 (PAD4) or Senescence-Associated Gene 101 (SAG101). TIR-encoded nicotinamide adenine dinucleotide hydrolase (NADase) produces signaling molecules to promote exclusive EDS1-PAD4 and EDS1-SAG101 interactions with helper NLR subclasses. In this work, we show that TIR-containing proteins catalyze adenosine diphosphate (ADP)-ribosylation of adenosine triphosphate (ATP) and ADP ribose (ADPR) through ADPR polymerase-like and NADase activity, forming ADP-ribosylated ATP (ADPr-ATP) and ADPr-ADPR (di-ADPR), respectively. Specific binding of ADPr-ATP or di-ADPR allosterically promotes EDS1-SAG101 interaction with helper NLR N requirement gene 1A (NRG1A) in vitro and in planta. Our data reveal an enzymatic activity of TIRs that enables specific activation of the EDS1-SAG101-NRG1 immunity branch.
Asunto(s)
ADP-Ribosilación , Adenosina Difosfato , Proteínas de Arabidopsis , Arabidopsis , Hidrolasas de Éster Carboxílico , Proteínas de Unión al ADN , Péptidos y Proteínas de Señalización Intracelular , Inmunidad de la Planta , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Arabidopsis/enzimología , Arabidopsis/inmunología , Proteínas de Arabidopsis/metabolismo , Hidrolasas de Éster Carboxílico/química , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Proteínas de Unión al ADN/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , NAD+ Nucleosidasa/metabolismoRESUMEN
Plant nucleotide-binding leucine-rich repeat-containing (NLR) receptors with an N-terminal Toll/interleukin-1 receptor (TIR) domain sense pathogen effectors to enable TIR-encoded nicotinamide adenine dinucleotide hydrolase (NADase) activity for immune signaling. TIR-NLR signaling requires the helper NLRs N requirement gene 1 (NRG1), Activated Disease Resistance 1 (ADR1), and Enhanced Disease Susceptibility 1 (EDS1), which forms a heterodimer with each of its paralogs Phytoalexin Deficient 4 (PAD4) and Senescence-Associated Gene 101 (SAG101). Here, we show that TIR-containing proteins catalyze the production of 2'-(5''-phosphoribosyl)-5'-adenosine monophosphate (pRib-AMP) and diphosphate (pRib-ADP) in vitro and in planta. Biochemical and structural data demonstrate that EDS1-PAD4 is a receptor complex for pRib-AMP and pRib-ADP, which allosterically promote EDS1-PAD4 interaction with ADR1-L1 but not NRG1A. Our study identifies TIR-catalyzed pRib-AMP and pRib-ADP as a missing link in TIR signaling through EDS1-PAD4 and as likely second messengers for plant immunity.
