Poster Presentation 49th Lorne Conference on Protein Structure and Function 2024

NMR, LCMS and XL-MS to characterise enzymatic activity of plant TIR domains in innate immunity (#206)

Mitchell Sorbello 1 , Bryan Ying Jie Lim 1 , Dalton Heng Yong Ngu 1 , Natsumi Maruta 1 , Jian Chen 2 , Mehdi Mobli 3 , Bostjan Kobe 1
  1. School of Chemistry and Molecular Biosciences and Australian Infection Diseases Research Centre, University of Queensland, Brisbane, QLD, Australia
  2. CSIRO Agriculture and Food, Canberra, ACT, Australia
  3. Centre for Advanced Imaging, Australian Institute for Bioengineering & Nanotechnology, The University of Queensland, St Lucia , QLD, Australia

Plant nucleotide-binding leucine-rich repeat immune receptors (NLRs) are activated by virulence factors (effector proteins) secreted inside plant cells by pathogens. NLRs consist of two regulatory domains which mediate effector recognition and NLR oligomerisation and a varying third N-terminal signalling domain. Toll-interleukin-1 receptor (TIR) domain containing NLRs (TNLs) posses’ NAD+ hydrolysis activity. Hydrolysis of NAD+ leads to production of a variety of compounds which activate downstream immune signalling pathways. Currently, it is unknown whether all TNLs produce the same compounds to initiate signalling. Additionally unpublished data suggests TIR domains may be able to interact with downstream signalling proteins EDS1 and the related proteins SAG101 and PAD4. In this work we aim to investigate the variability of plant TIR enzymatic activity using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and nuclear magnetic resonance (NMR). We also aim to detect potential TIR-EDS1 interactions in vitro using cross-linking mass spectrometry (XL-MS) and electron microscopy. Optimisation of XL-MS protocol is still ongoing; however, our results indicate that plant TIRs do vary in the products they produce which may highlight alternate catalytic mechanisms or signalling pathways within the cell.