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 di-ADPR or ADPr-ATP 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.

Chemical structures of cyclic ADP ribose (cADPR) isomers and the molecular basis of their production and signaling

“…The cleavage of the pyrophosphate bond could indicate the involvement of plant NUDIX hydrolases like NUDX6/7, which regulate plant immunity by degrading 2'3'-cAMP/2'3'-cGMP, the other nucleotides putatively produced by TIR domains (41). 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) (46). Interestingly, 3′-O-β-D-ribofuranosyladenosine, which has an identical 1''-3' O-glycosidic linkage to v2-cADPR (3'cADPR) but lacks the phosphate groups, has been shown to accumulate in leaves infected with the HopAM1 producing bacterium Pseudomonas syringae DC3000 (62), suggesting that cADPR isomers can be further modified in plants.…”

“…Bacterial and plant TIR domains produce cyclic signaling nucleotides with immune and virulence functions, using NAD + or nucleic acids as substrate ( 26, 27, 33, 34, 41, 46 ). 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 cleavage of the pyrophosphate bond could indicate the involvement of plant NUDIX hydrolases like NUDX6/7, which regulate plant immunity by degrading 2’3’-cAMP/2’3’-cGMP, the other nucleotides putatively produced by TIR domains ( 41 ). 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) ( 46 ).…”

Pangenomic analysis reveals plant NAD+manipulation as an important virulence activity of bacterial pathogen effectors

“…The structure of v-cADPR was recently identified as an isomer of cADPR and renamed 2’-cADPR (Manik et al, 2022). Other TIR generated products pRib-AMP/ADP, ADPR-ATP and diADPR were shown to bind to and activate specific EDS1 heterodimers (Huang et al, 2022; Jia et al, 2022), indicating these small molecules link TIR activation to EDS1-dependant immune signalling. In addition, TIR-only proteins also exhibit 2’,3’-cNMP synthase activity in vitro and these cyclic nucleotides may also contribute to immunity (Yu et al, 2022).…”

“…TIR-NLR-triggered immunity is usually dependant on downstream Enhanced Disease Susceptibility 1 (EDS1)-helper NLR pathways (Adlung et al, 2016). Plant TIRs produce various products, including ADPR, NAM and a v-cADPR (Table 1), through NAD + hydrolysis (Huang et al, 2022; Jia et al, 2022; Ma et al, 2020; Wan et al, 2019). The structure of v-cADPR was recently identified as an isomer of cADPR and renamed 2’-cADPR (Manik et al, 2022).…”