Probing the oligomeric state of activated NLR immune receptors by blue native-polyacrylamide gel electrophoresis (BN-PAGE)

*This is a poster for exhibition in IS-MPMI 2023

Nucleotide-binding domain leucine-rich repeat (NLR) proteins play a vital role in plant and metazoan innate immunity, oligomerizing into multiprotein complexes known as resistosomes or inflammasomes upon activation. NLRs can function as individual singletons or in higher-order configurations, such as pairs, or networks, which exhibit a sensor-helper dynamic. While recent structural studies have provided ground-breaking insights into the molecular mechanisms of singleton NLR activation, the biochemical mechanisms underlying the activation of paired and networked NLRs remain unclear. In this study, we leveraged blue native polyacrylamide gel electrophoresis (BN-PAGE) to investigate NLR activation, focusing on the Pik1/Pik2 system and the NRC (NLR required for cell death) network as models for paired and networked NLRs, respectively. We found that the engineered Pikm-1/Pikm-2 sensor-helper pair (aka Pikobodies) constitutively form an oligomeric hetero-complex, which incorporates its cognate effector for activation. In contrast, the sensor/helper pairs in the NRC network follow an activation-and-release model, in which sensor NLRs mediate the formation of helper NRC homo-oligomers upon effector perception. We will introduce the method pipeline for establishing BN-PAGE assays for studying NLR biology and discuss our results, which highlight BN-PAGE as a valuable and versatile tool for visualizing NLR activation and studying oligomeric resistosomes and their components. Our findings contribute to a better understanding of the molecular mechanisms underlying plant innate immunity and may have implications for the development of novel plant disease resistance strategies.

Keywords: NLR resistosome, NLR activation, Blue Native PAGE