Investigation of the recognition and activation of plant NLRs
Creators: name: Chen, Jian
Publisher: name: The Australian National University
Year: 2022

description: Plants utilize intracellular TIR-NB-LRR (TNL) and CC-NB-LRR (CNL) immune receptors to recognise pathogen effectors, causing effector-triggered immunity (ETI). While increasing evidence support the CC domain of CNL proteins can execute the immune response directly by forming membrane-penetrating channels, the TIR domain of TNL proteins requires further downstream signalling partners to trigger the defense response. A major purpose of this thesis is to better understand the signalling function of TIR domains and the downstream signalling events that occur after TNL activation. In chapter 2, I showed that an intrinsic NADase catalytic activity of plant TIR domains is indispensable for downstream signalling activation by analysis of TIR domain mutants with both loss and gain of activity phenotypes. In addition, I showed that self-association is a sufficient precondition for the signalling function of TIR domain. There are two layers of signalling partners operating downstream of TIR domains: the EDS1 family heterodimers EDS1-PAD4 and EDS1-SAG101, and the helper CNLs, ADR1 and NRG1. It has been proposed that cell death triggered in N. benthamiana by some TNLs only requires NbEDS1, NbSAG101b and NbNRG1. In chapter 3, I tested 16 different TIR containing constructs, which cause cell death in wildtype N. benthamiana, in gene knockout plants that lack various members of the EDS1 family or NRG1. The results showed that for TIRs and TNLs mediated cell death, NbEDS1, NbSAG101b and NbNRG1 are commonly required, while NbPAD4 and NbSAG101a are not required. Then CoIP and split-Luciferase assays showed that plant TIRs physically interact with the EDS1 family proteins in planta, mainly via their N-terminal lipase-like domains. A yeast three hybrid analysis showed plant TIRs also interact with the NbEDS1-NbSAG101b heterodimer. These data suggest that plant TIR signalling through EDS1/SAG101b involves direct interaction between the proteins, possibly facilitating transfer of a signalling molecule generated by their NADase activity. In chapter 4, I investigated why NbSAG101b is required, but NbSAG101a and NbPAD4 are not required for the cell death triggered by TNLs. Using CoIP, I found that only NbSAG101b interacts with NbNRG1, which is mainly mediated through the C-terminal EP domain of SAG101b interacting with the NB and LRR domains of NbNRG1. Domain swaps between NbSAG101b and NbSAG101a or NbPAD4 indicated chimeras contain the C-terminal domain of NbSAG101b can also interact with NbNRG1 and functionally mediate TNL triggered cell death. In summary, these data lead me to propose that in N. benthamiana plant TIR domains interact with the N-terminal domain of EDS1 heterodimers, which likely facilitates transfer of NADase catalytic products to the complex. Changes in the conformation of EDS1 heterodimers enable the C-terminal domain of NbSAG101b to interact with the NB and LRR domains of NbNRG1 and activate it from an inhibited state. Pathogen effectors play crucial roles in suppressing plant immunity and can also be monitored by NLR proteins to trigger immune response. In chapter 5, I examined specific recognition events between a wheat NLR protein, Sr50, and AvrSr50. Through mutational analysis of recognised and non-recognised alleles of AvrSr50, I identified a single polymorphic residue that determines the difference in their recognition in both in planta functional assays and in yeast-two-hybrid protein interaction. I also tested a variety of other newly identified AvrSr50 allelic variants and assigned them as either recognised or non-recognised alleles. This analysis provides necessary information for predicting virulence phenotypes of novel strains based on genome sequence alone. In summary, the work presented in this thesis has improved our understanding of the mechanisms involved in plant NLRs induction of defense responses. This could help crop breeders to develop new strategies to obtain broad and durable resistance to pathogens.