Immunomodulatory purine nucleotides, not NAD+ depletion, are associated with TIR domain-mediated cell death

Toll/interleukin-1 receptor (TIR) domain proteins activate or suppress plant immunity by generating immunomodulatory purine nucleotides (IPNs) from NAD⁺. Plant TIR-domains convert NAD⁺ into a range of IPNs including ADPR and 2'cADPR while promoting immune activation and plant cell death. Conversely, the phytopathogenic TIR-domain effector HopAM1 converts NAD⁺ into 3'cADPR while suppressing plant immunity. However, it is still unclear if NAD⁺ depletion plays a significant role in TIR-mediated cell death caused by HopAM1 or plant TIR-domains. HopAM1 induces moderate accumulation of an unidentified molecule in Arabidopsis thaliana Col-0 and Xan-2. HopAM1’s TIR domain alone (HopAM1_TIR) generates cADPR, ADPR, and an additional unidentified product in vitro rather than 3'cADPR. Pseudomonas fluorescens (pHIR11)-delivered HopA1₆₁ induces ADPR in Nicotiana tabacum cv. Xanthi but cADPR in A. thaliana Ws-0. HopAM1 suppresses production of both IPNs while blocking cell death. HopA1₆₁-induced cell death in N. tabacum cv. Xanthi correlates with production of IPNs, not with a depletion of NAD⁺. Finally, the IPN-binding phage protein Tad1 (Thoeris anti-defense 1) blocks HopAM1-induced cell death in yeast. Our results show considerably more complexity to TIR-domain enzymatic activity and indicate that NAD⁺-depletion is unlikely to be the primary mechanism of either HopAM1 or plant TIR domain-mediated cell death.