Plant pathogens constitute a major threat to global food security. The use of naturally resistant crop varieties can limit crop losses, however new races of pathogen can arise that are able to overcome these defences. Plant breeding for race-specific resistance typically relies on disease-resistance genes, which generally encode proteins with nucleotide-binding and leucine-rich repeat domains (NB-LRRs). NB-LRRs are a large of proteins found in both plants and animals, with plant NB-LRRs further classified by the presence of N-terminal coiled-coil or toll-interleukin receptor domains. Although qualitative models exist to describe R-protein regulation and activation, these are predominantly based on genetic and molecular studies. Biochemical investigations into R-protein function have been hampered by difficulties obtaining sufficient yields of material. When suitable material has been identified, biochemical studies have been used to complement well-established in planta assays to validate numerous hypotheses. This work describes the screening processes undertaken to obtain R-protein domains suitable for downstream experiments. Using E. coli for high-throughput screening of constructs from a single R-protein, traditional construct design to investigate multiple R-protein domains and expanding our expression hosts to eukaryotic systems we successfully purified four coiled-coil domains and a single NBARC domain for use in downstream experiments. Characterisation of this NBARC domain by circular dichroism and small-angle X-ray scattering indicates that the protein is well-folded and stable in solution, allowing in vitro investigations. In testing models for R-protein regulation we were able to confirm previous findings, such as low levels of ATPase activity, however we were unable to find evidence for a commonly cited method of signal repression. A preliminary crystal structure of the NBARC domain shows significant similarity to Apaf-1, and highlights the importance of conserved motifs in NBARC architecture. The tools presented here should prove a valuable resource to complement existing models to better understand the structure, biochemistry, and ultimately regulation of plant R-proteins.
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