TIR-catalyzed ADP-ribosylation reactions produce signaling molecules for plant immunity

Abstract

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 NADases produce signaling molecules to promote exclusive EDS1-PAD4 and EDS1-SAG101 interactions with helper NLR sub-classes. Here we show that TIR-containing proteins catalyze adenosine diphosphate (ADP)-ribosylation of adenosine triphosphate (ATP) and ADP ribose (ADPR) via 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.

Cyclic ADP ribose isomers: Production, chemical structures, and immune signaling

“…Bacterial and plant TIR domains produce cyclic signaling nucleotides with immune and virulence functions using NAD + or nucleic acids as substrates ( 14 , 15 , 21 , 22 , 26 , 32 , 33 , 38 – 40 ). Here, we report the chemical structures of two TIR domain–produced cADPR isomers, v-cADPR and v2-cADPR, which reveal that TIR domains can catalyze O-glycosidic bond formation between the ribose sugars in ADPR and that cyclization occurs at the 2′ (v-cADPR; 2′cADPR) and 3′ (v2-cADPR; 3′cADPR) positions of the adenosine ribose.…”

“…The nucleotides pRib-AMP and pRib-ADP [2′-(5″-phosphoribosyl)-5′-adenosine monophosphate and 2′-(5″-phosphoribosyl)-5′-adenosine diphosphate] were shown to trigger immune signaling in plants by allosterically promoting the EDS1 (enhanced disease susceptibility 1)–PAD4 (phytoalexin deficient 4) complex to bind to the plant NLR protein ADR1-L1; the production requires TIR proteins ( 32 ). Plant TIR domains can also generate ADP-ribosylated ATP (ADPr-ATP) and di-ADPR, which in turn promote the association of EDS1 and SAG101 (senescence-associated gene 101) with the helper NLR NRG1A (N requirement gene 1A) ( 33 ).…”

“…Multiple TIR domains produce cADPR isomers in plants, but the EDS1-binding molecules have not yet been detected in planta and their biosynthetic pathways are not defined. The authors of ( 32 , 33 ) proposed a pathway for the production of pRib-AMP and pRib-ADP as well as ADPr-ATP and di-ADPR that does not involve cADPR isomers. Our cADPR isomer structures show that pRib-AMP can be derived directly from v-cADPR (2′cADPR) by cleavage of its pyrophosphate bond, suggesting that NAD + could be the substrate in this case.…”

“…Bacterial and plant TIR domains produce cyclic signaling nucleotides with immune and virulence functions using NAD + or nucleic acids as substrates (14,15,21,22,26,32,33,(38)(39)(40).…”

“…S2B). We also tested the activities in the presence of NAD + , adenosine triphosphate (ATP), and Mg 2+ and observed no formation of metabolites reported for plant TIR domains (32,33) (fig. S1F).…”