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

Abstract

Cyclic ADP ribose (cADPR) isomers are important signaling molecules produced by bacterial and plant Toll/interleukin-1 receptor (TIR) domains via NAD+ hydrolysis, yet their chemical structures are unknown. We show that v-cADPR (2’cADPR) and v2-cADPR (3’cADPR) isomers are cyclized by O-glycosidic bond formation between the ribose moieties in ADPR. Structures of v-cADPR (2’cADPR)-producing TIR domains reveal that conformational changes are required for the formation of the active assembly that resembles those of Toll-like receptor adaptor TIR domains, and mutagenesis data demonstrate that a conserved tryptophan is essential for cyclization. We show that v2-cADPR (3’cADPR) is a potent activator of ThsA effector proteins from Thoeris anti-phage defence systems and is responsible for suppression of plant immunity by the effector HopAM1. Collectively, our results define new enzymatic activities of TIR domains, reveal the molecular basis of cADPR isomer production, and establish v2-cADPR (3’cADPR) as an antiviral signaling molecule and an effector-mediated signaling molecule for plant immunity suppression.One-Sentence SummaryThe chemical structures of two O-glycosidic bond-containing cyclic ADP ribose isomers, the molecular basis of their production, and their function in antiviral and plant immunity suppression by bacteria are reported.

Activation and Regulation of NLR Immune Receptor Networks

“…TIR-NLRs thus indirectly induce a similar immune response to the CC-NLRs. The likely structure of plant TIR-domain produced v-cADPR was recently identified as 2'cADPR (independently identified and named 1′-2′ glycocyclic ADPR [gcADPR]) (Manik et al, 2022;Leavitt et al, 2022). 2'cADPR may serve as an intermediate in the synthesis of novel nucleosides associated with plant immunity (Manik et al, 2022).…”

“…The likely structure of plant TIR-domain produced v-cADPR was recently identified as 2'cADPR (independently identified and named 1′-2′ glycocyclic ADPR [gcADPR]) (Manik et al, 2022;Leavitt et al, 2022). 2'cADPR may serve as an intermediate in the synthesis of novel nucleosides associated with plant immunity (Manik et al, 2022). Indeed, Huang et al, (2022) show that TIR-NLRs catalyse the production of 2'-(5''-phosphoribosyl)-5'-adenosine mono-/diphosphate (pRib-AMP/ADP), and that EDS1-PAD4 act as a receptor complex for pRib-AMP/ADP.…”

“…Binding of pRib-ADP to EDS1-PAD4 results in a conformational change in the PAD4 C-terminal domain, thereby promoting interaction with ADR1s (Huang et al, 2022). pRib-AMP can be directly derived from 2'cADPR by cleavage of its pyrophosphate bond (Manik et al, 2022). EDS1-SAG101, instead, acts as a receptor complex for other TIRcatalysed second messengers, ADP-ribosylated ATP (ADPr-ATP) and ADPr-ADPR (di-ADPR), that induce EDS1-SAG101 association with NRG1.1 but not with ADR1-L1 .…”

“…Similarly, the AvrA1 effector from the bacterial pathogen Pseudomonas syringae interacts with the soybean (Glycine max) homologs of EDS1, and requires these proteins to exert its virulence function (Wang et al, 2014). Finally, the P. syringae effector HopAM1 is a TIR-domain containing effector which can suppress cellsurface-mediated signalling and TIR-NLR mediated signalling (Eastman et al, 2022;Manik et al, 2022). HopAM1 serves to produce a distinct version of v-cADPR recently identified as 3'cADPR (Manik et al, 2022).…”

“…Finally, the P. syringae effector HopAM1 is a TIR-domain containing effector which can suppress cellsurface-mediated signalling and TIR-NLR mediated signalling (Eastman et al, 2022;Manik et al, 2022). HopAM1 serves to produce a distinct version of v-cADPR recently identified as 3'cADPR (Manik et al, 2022). It appears that 3'cADPR and its derivatives can manipulate ADR1/NRG1 network signalling (Manik et al, 2022).…”