Poster Presentation 50th Lorne Proteins Conference 2025

Structural and functional characterisation of the flax L6 innate immune receptor (#419)

Dalton H. Y. Ngu 1 2 3 , Natsumi Maruta 1 2 3 , Bryan Y. J. Lim 1 2 3 , Mtichell J. Sorbello 1 2 3 , Hayden L. Burdett 4 , Bostjan Kobe 1 2 3
  1. School of Chemistry & Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia
  2. Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, Queensland, Australia
  3. Institute for Molecular Bioscience, The University of Queesland, Brisbane, Queensland, Australia
  4. Wellcome Centre for Cell Biology, The University of Edinburgh, Edinburgh, EH9 3BF, Scotland, The United Kingdom

In plant innate immunity, specialised intracellular receptors known as NLRs (nucleotide-binding leucine-rich repeat receptors), recognise secreted effector proteins from pathogenic microbes and activate downstream defence responses. One major group of NLRs is TNLs (TIR-NLRs), whose domain architecture consists of an N-terminal TIR (Toll/interleukin-1 receptor) signalling domain, a central NB-ARC (nucleotide-binding adaptor, APAF-1, R proteins and CED-4) domain, and a C-terminal LRR (leucine-rich repeat) domain. Upon effector binding, TNLs oligomerise to form tetrameric protein complexes called resistosomes. Via the TIR domains, these TNL resistosomes possess NAD+ (nicotinamide adenine dinucleotide) hydrolase activity to produce a wide range of metabolic and signalling molecules, such as 2’cADPR (variant cyclic adenosine diphosphate ribose), triggering downstream signalling and leading to basal immune response or localised cell death to prevent pathogen spread. In addition, a recent study has shown that some TIR domains from plants, including the flax TNL L7TIR, can bind DNA and RNA to form large filamentous assemblies, which exhibit nucleotide cleavage activity and produce 2’,3’-cAMP/cGMP. Given that the TNL-mediated immune signalling mechanism is still unclear, this study aims to structurally and functionally characterise the flax TNL L6 protein that is known to mount defence signalling against the flax rust fungus Melampsora Lini. We successfully expressed and purified both the L6TIR domain and the full-length L6 protein and found that they exhibit NADase activity to produce 2’cADPR, with the full-length protein exhibiting stronger enzymatic activity upon incubation with its pathogen effector, AvrL567-A. Like L7TIR, we found that L6TIR also formed filamentous assemblies upon DNA and RNA binding, and we determined its filamentous structure by cryo-electron microscopy. In our current and future work, we aim to establish if L6 forms a tetrameric resistosome, and to characterise metabolites produced by L6 upon NADase activity and DNA/RNA cleavage.