Flax NLRs and the Equilibrium-Switch Model

Author: Hayden Burdett

Burdett, Hayden, 2018 Flax NLRs and the Equilibrium-Switch Model, Flinders University, College of Science and Engineering

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Plants are susceptible to pests and disease. Unlike animals, each plant cell is required to defend itself against pathogen invasion. A two-tiered innate immune system allows plants to combat pathogens. The role of the first tier to deter common, non-specialised pathogens from infecting the host, whist the role of the second tier is to defend against pathogens able to bypass the first, which pathogens achieve by secretion of effector proteins.

Plants can detect and respond to secreted effectors through proteins known as Nucleotide Binding (NB) Leucine-Rich Repeat (LRR) domain Receptors (NLRs). Plant NLRs induce a robust immune response, characterised by localised cell death around the site of infection, known as a hypersensitive response. Despite extensive work, the mechanism by which plant NLRs are regulated, how they perceive effectors, and how they signal a hypersensitive response are still unclear.

These questions can be probed by examination of the full-length plant NLRs in vitro, which, to date, has proven difficult. Here, the nucleotide binding of L6 and L7, NLRs from flax purified from Pichia pastoris, are reported. Flax NLRs able to induce effector-dependent cell death were found to be bound with low levels of ADP, whilst those that induced a weaker, slower immune response were found to be bound with high levels of ADP. Previous published data demonstrated that other auto-active plant NLRs preferentially bind ATP over ADP. Collectively, these data, combined the results of with in planta and yeast-2-hybrid assays on L6, L7 and mutants created by collaborators, suggests an equilibrium-switch model of activation, where a plant NLR cycles between ADP/ATP bound states, only detecting an effector when in an ATP-bound state.

To further interrogate this model NLRs protein of higher purity and yield were required. Modification of existing techniques allowed for increased purity and yields of three flax NLRs. Using StrepTactin Affinity Chromatography, M, L6 and L7 were purified to >90% purity, whilst maintaining yields suitable for both biochemical and biophysical experiments.

Purified M, AvrM and avrM proteins was used in the analysis of the interaction between a full-length NLR and effector in vitro, utilising Surface Plasmon Resonance (SPR). The results showed that M interacts weakly with AvrM in vitro, but that no interaction was detected between M and avrM. The interaction of M with various gain-of-function and loss-of-function mutants of AvrM and avrM was also investigated, but interaction kinetics were not able to be calculated.

The results presented answer questions about the activation, and interaction with effectors, of flax NLRs. However, how the difference in nucleotide binding of flax NLRs influences their interaction with their cognate effectors remains to be answered. Improved purification of plant NLRs will enable more diverse experiments to be conducted, such as the structural characterisation of full-length plant NLRs, and further analysis of NLR/effector interaction in the presence and absence of nucleotides using SPR. These experiments will be critical if we are to completely unravel the function of these important class of plant proteins.

Keywords: Plant Innate Immunity, NLRs, Flax, Flax Rust, Proteins, R proteins

Subject: Biological Sciences thesis

Thesis type: Doctor of Philosophy
Completed: 2018
School: College of Science and Engineering
Supervisor: Peter Anderson