Structural and Biochemical Analysis of Plant TIR NAD plus Cleavage Activity [Meeting Abstract]

Plant NLRs (Nucleotide binding, Leucine-rich repeat Receptors) provide resistance to a range of biotrophic pathogens, by recognition of avirulence factors, and proceeded by initiation of downstream immune responses. NLR signaling is transduced through the N-terminal TIR domain, and resistance is often characterized by localized cell death around the site of infection. How plant TIR domains transduce this signal is still unknown, however, like TIR domains from mammalian NLRs, plant TIR domains form homo- and heterotypic dimers, and this dimerization is required for signaling. Recently, a TIR domain from the mammalian TLR adaptor family, SARM1, was shown to possess NADase activity, the first report of a TIR domain displaying enzymatic activity. Given the structural similarities between SARM TIR and plant TIR domains, the requirement of SARM dimerization for NADase activity, and the lack of downstream binding partners identified for plant NLRs, we hypothesize that some plant TIR domains also possess NADase activity. Here we present biochemical and structural evidence that plant TIR domains possess NADase activity, and new insights into how this activity is involved in eliciting HR. NAD+ is cleaved into ADPR and Nicotinamide in vitro by the TIR domains from RUN1 and L6. NADase activity can be increased by addition of macromolecular crowding agents. Mutations to the NADP+ binding site in the RUN1 structure also effect NADase activity.