Pathogens translocate virulence proteins known as effectors into plant cells to target specific host proteins and establish a better intracellular environment for infection. Plants have in turn developed effector triggered immunity encompassing a sophisticated network of immune receptors, including the nucleotide binding, leucine rich repeat (NLR) family. These receptors can recognise non-self proteins, such as pathogen effectors, and in response relay a signal to initiate an immune response. Most NLRs from cereals have a conserved chassis, comprised of a CC-domain, an NB-ARC domain, and a LRR-domain. However, some sensor NLRs contain a non-canonical domain that has been integrated into the chassis to act as a decoy to detect the presence of pathogen effectors. The binding of an effector to the non-canonical domain most likely leads to a conformational change in the sensor NLR, which then initiates immune signalling in the combination with the paired helper NLR.
The Mark Banfield lab at the John Innes centre, in which my project was based, focusses on biochemical and structural properties of the Pik NLR allelic series from Oryzae sativa in which the sensor NLR of the Pik-1 and Pik-2 pair uses an integrated non-canonical heavy metal-associated (HMA) domain as a decoy against the corresponding avirulence effectors of Magnaporthe oryzae (AVR-Pik allelic series). Magnaporthe oryzae is the biggest pre-harvest biotic killer of rice, which is the staple crop for half the world’s population.
Previous work identified that variation in the HMA domain determines the specificity of binding to AVR alleles and has supported the design of amino acid mutations in the HMA domain of the Pikp-1 allele to extend recognition across AVR-Pik allelic series. Pikp-1 HMA was mutated to contain a single amino acid change in the central binding interface with the effectors (mimicking the HMA of Pikm-1 NLR allele), and further amino acid substitutions were added in the C-terminal binding interface that had previously been shown to be involved in extending recognition. I successfully made mutated constructs and carried out a hypersensitive response-based assay using Agrobacterium-mediated infiltration with these resources. Unfortunately, time restraints meant only preliminary results from this assay were obtained.
Alongside this, structural homology modelling combined with sequence alignments identified a region in the NB-ARC domain of the Pik-2 helper NLR that is not present in other NLR NB-ARC domains. This region in Pik-2 may have appeared at a similar time to the integration of the HMA domain in Pik-1 in evolutionary time. We hypothesised that this region may be involved in Pik-2 sensing of the biochemical status of Pik-1, and could be important for initiation of downstream immune signalling post-effector binding. In this project I used a mutant deleting this region, and completed cell death assays through Agrobacterium infiltration of N. benthamiana to investigate the consequences of removal. The results of the assays indicate the ability of the Pik NLR pair to signal is compromised when the region is removed, as the known cell death is lost against both the effector as well as in Pikp-1 and Pikp-2 auto-active mutants. This acts as initial evidence supporting the hypothesis that this region is important for Pik receptor function, and further that the Pik pair may signal in a complex. However, further work must be done to demonstrate that the loss of cell death is not an artefact of motif removal leading to a misfolding and non-functional protein.
The 8 week project sponsored by the BSPP has been a fantastic opportunity that has given me the confidence to consider a PhD and enabled me to discover a very important and intriguing subject in plant pathology.
University of Glasgow