This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
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This summer I spent 8 weeks in Mark Banfield’s lab at the John Innes Centre characterising effector perception by a novel immune receptor. The Banfield lab’s focus is on plant pathogen interactions, focusing on the structure and function of nucleotide-binding, leucine-rich repeat receptor proteins (NLRs), which are intracellular immune receptors in plants. The research done by the lab aims to guide the engineering of disease resistant crops. The NLR I was studying is known to recognise several effectors with a shared structural fold. The main part of my project involved testing whether this NLR can recognise a broad range of effectors with this fold, from different species. To test this, I used cell death assays in Nicotiana benthamiana. My project began using golden gate cloning to place the effector genes into plasmids. These plasmids containing the genes of interest were transformed into agrobacterium, which was infiltrated into the leaves of N. benthamiana. This allows agrobacterium-mediated transient expression of the genes of interest in the infiltrated spots on the leaves. When an NLR is infiltrated alongside an effector it recognises, this causes an immune response by the plant, causing visible cell death. From these cell death assays, I found that the NLR under investigation could recognise effectors carrying the same fold to varying degrees, some were recognised strongly, and others not at all.
Alongside the cell death assays, I also investigated the possibility of purifying the NLR from expression in Escherichia coli. This began by testing whether the NLR was able to be expressed in E. coli, by adding a plasmid with the gene of interest to E. coli, then using detergent to lyse the cells. The lysate was then run on an SDS-PAGE gel, to see if the protein of interest could be detected. Next, a sonication test was run to compare protein presence in the soluble vs. insoluble parts of the cell. I compared the level of expression of the NLR with a range of tags in several different E. coli strains. The strain and tag combination which showed best expression of soluble protein was then selected to be used for a benchtop purification. Purification was attempted using affinity chromatography based on the tags selected. The work started by this project, as well as the wider research of the Banfield group, has implications for the design of NLRs with increased disease resistance, in particular against wheat blast and rice blast disease, which are major threats against global agriculture.
This summer project has given me the opportunity to experience what it’s like to work in plant pathology, teaching me a range of laboratory skills and giving me the confidence to continue my studies in plant sciences after my undergraduate degree. I would like to thank the BSPP for funding me to be able to carry out this project, as well as the Banfield lab for hosting me, and especially Clemence Rodney for her brilliant day to day supervision.
Mia Cohen
University of Oxford
