Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors activate cell death and confer disease resistance by unknown mechanisms. We demonstrate that plant Toll/interleukin-1 receptor (TIR) domains of NLRs are enzymes capable of degrading nicotinamide adenine dinucleotide in its oxidized form (NAD+). Both cell death induction and NAD+ cleavage activity of plant TIR domains require known self-association interfaces and a putative catalytic glutamic acid that is conserved in both bacterial TIR NAD+-cleaving enzymes (NADases) and the mammalian SARM1 (sterile alpha and TIR motif containing 1) NADase. We identify a variant of cyclic adenosine diphosphate ribose as a biomarker of TIR enzymatic activity. TIR enzymatic activity is induced by pathogen recognition and functions upstream of the genes enhanced disease susceptibility 1 (EDS1) and N requirement gene 1 (NRG1), which encode regulators required for TIR immune function. Thus, plant TIR-NLR receptors require NADase function to transduce recognition of pathogens into a cell death response.

Summary The Arabidopsis NB-LRR immune receptor RPM1 recognizes the Pseudomonas syringae type III effectors AvrB or AvrRpm1 to mount an immune response. Although neither effector is itself a kinase, AvrRpm1 and AvrB are known to target Arabidopsis RIN4, a negative regulator of basal plant defense, for phosphorylation. We show that RIN4 phosphorylation activates RPM1. RIN4142–176 is necessary, and with appropriate localization sequences, sufficient to support effector-triggered RPM1 activation, with the threonine residue at position 166 being critical. Phosphomimic substitutions at T166 cause effector-independent RPM1 activation. RIN4 T166 is phosphorylated in vivo in the presence of AvrB or AvrRpm1. RIN4 mutants that lose interaction with AvrB cannot be co-immunoprecipitated with RPM1. This defines a common interaction platform required for RPM1 activation by phosphorylated RIN4 in response to pathogenic effectors. Conservation of an analogous threonine across all RIN4-like proteins suggests a key function for this residue beyond the regulation of RPM1.

Plants deploy intracellular innate immune receptors to recognize pathogens and initiate disease resistance. These nucleotide-binding, leucine-rich repeat (NB-LRR) proteins are activated by pathogen effector proteins that are delivered into the host cell to suppress host defense responses. Little is known about the sites and mechanisms of NB-LRR activation, but some NB-LRR proteins can function inside the plant nucleus. We demonstrate that RPM1 is activated on the plasma membrane and does not relocalize to the nucleus. An autoactive RPM1(D505V) allele that recapitulates key features of normal RPM1 activation also resides on the plasma membrane. There is no detectable relocalization of activated RPM1 to the nucleus. Hindering potential nuclear entry of RPM1-Myc did not affect either its effector-triggered hypersensitive-response (HR) cell death or its disease resistance functions, further suggesting that nuclear translocation is not required for RPM1 function. RPM1 tethered onto the plasma membrane with a dual acylated N-terminal epitope tag retained the ability to mediate HR, consistent with this RPM1 function being activated on the plasma membrane. Plant NB-LRR proteins can thus function at various locations in the cell.

Detection of pathogens by plants is mediated by intracellular nucleotide-binding site leucine-rich repeat (NLR) receptor proteins. NLR proteins are defined by their stereotypical multidomain structure: an N-terminal Toll-interleukin receptor (TIR) or coiled-coil (CC) domain, a central nucleotide-binding (NB) domain, and a C-terminal leucine-rich repeat (LRR). The plant innate immune system contains a limited NLR repertoire that functions to recognize all potential pathogens. We isolated Response to the bacterial type III effector protein HopBA1 (RBA1), a gene that encodes a TIR-only protein lacking all other canonical NLR domains. RBA1 is sufficient to trigger cell death in response to HopBA1. We generated a crystal structure for HopBA1 and found that it has similarity to a class of proteins that includes esterases, the hemebinding protein ChaN, and an uncharacterized domain of Pasteurella multocida toxin. Self-association, coimmunoprecipitation with HopBA1, and function of RBA1 require two previously identified TIR-TIR dimerization interfaces. Although previously described as distinct in other TIR proteins, in RBA1 neither of these interfaces is sufficient when the other is disrupted. These data suggest that oligomerization of RBA1 is required for function. Our identification of RBA1 demonstrates that "truncated" NLRs can function as pathogen sensors, expanding our understanding of both receptor architecture and the mechanism of activation in the plant immune system. plant immunity | NLR | Toll-interleukin-1 receptor homology domain | oligomerization | type III secretion