Although genetic modification of plants remains a hot topic of discussion, genetic engineering of disease resistant crops is one of the most promising ways of combating pre-harvest yield losses caused by pests and diseases.
My summer project in Mark Banfield’s lab at the John Innes Centre was aimed at engineering novel effector recognition into the Pik NLR (Nucleotide Binding, Leucine-rich repeat) receptor from rice. In nature, the Pik NLR confers resistance in rice against strains of the fungal pathogen Magnaporthe oryzae that express the AVR-Pik effector. Using the golden gate modular cloning system, I cloned various protein domains into a Pik NLR chassis, and was responsible for cloning cognate pathogen effector proteins into level 1 acceptor vectors for plant expression. Then, using agrobacterium mediated infiltration, I conducted immunity-related cell death assays in Nicotiana benthamiana, testing for both the potential for autoactivity of the engineered NLRs and for pathogen effector recognition.
Of the 6 protein domains I successfully cloned and tested in the short timeframe of the project, one delivered very promising results. This engineered Pik NLR chassis was, in this case, not auto active, but when co-infiltrated with an effector previously shown to bind the incorporated protein domain, cell death was observed. This indicates that the engineered NLR was responding to the effector. Further work is now being progressed in the lab to investigate the efficacy of this engineered NLR and its specificity of binding, development of which presents very exciting perspective into plant immunity. This project represents an exciting proof of concept that new effector recognition specificities can be engineered into the Pik NLR chassis, using unrelated integrated domains.
During the project, a second aim was to try and correlate observed cell death with interaction between engineered NLRs and effectors by co-immunoprecipitation. Frustratingly, this specific part of the project was impacted by Covid-related complications during the final stages. At this time, I concentrated more on reading literature related to the project, and plant immunity more broadly. During the project, I really appreciated the opportunity to work in a new system and enjoyed the in planta work. Coming from a biomedical sciences background, this project gave great insights into plant work and more specifically plant pathology. I learnt that just like humans, plants are affected by diseases, and discovered how important it is that we find lasting genetic solutions to plant diseases, to ensure food security and limit the damaging impact of agriculture on the environment.
Brenda Kageni Mionki
Above: Localised cell death in Nicotiana benthamiana at the site of NLR and effector infiltration. This suggests that the NLR is recognising the effector and triggering the immune system causing the localised cell death.