Written by Karen Irawan.
This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
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Fusarium graminearum (Fg) is a fungal pathogen of wheat and the main causal agent of the devastating Fusarium head blight (FHB) disease. FHB is associated with the production of mycotoxins that make the harvested grains harmful to humans and farmed animals, resulting in major economic losses. Pathogens such as Fg secrete effectors are proteins that help to facilitate infection by suppressing or evading the host’s immune response. Research on Fg effector proteins and their functions gives us a better understanding of the mechanisms underlying infection, which is essential in designing FHB-resistant wheat.
At Rothamsted Research, my project explored the effects of putative Fg effectors on the plant immune response. I wanted to find out whether the putative effectors could suppress cell death induced by BAX and INF1, as an important function of the immune system is to rapidly kill infected cells and thereby slowing the spread of infection. The putative effectors investigated are upregulated in the presymptomatic phase of Fg infection, which I confirmed using qPCR. Their effects on cell death were investigated using Agrobacterium-mediated transient expression in model plant Nicotiana benthamiana. This involves cloning an effector into a pEAQ plasmid, transforming the plasmid into Agrobacterium tumefaciens, and infecting N. benthamiana. The effector gene is then integrated into the plant’s chromosome and transiently expressed at high levels. The induced cell death levels in infected leaves were then visualised using DAB staining, which indicates areas within the leaves with high levels of reactive oxygen species (ROS) production associated with stress. Cell death was also quantified by measuring electrolyte leakage from leaf cells. The project also included bioinformatics analysis of effector genes and upstream regions to find possible regulatory motifs.
The results obtained revealed possible interaction between two effector proteins, but there was high variability in the data. I found it challenging to analyse the results, which often brought up more questions, and I learnt to design better experiments over the course of the project. Hearing other perspectives showed me how to explore every possibility and think creatively. I gained valuable skills in problem solving and a lot of experience in the lab, which will be useful as I aim to pursue a career in research. This project has given me a better appreciation for crop research and its impact.
I am grateful to BSPP for this amazing opportunity. I would also like to thank Rothamsted Research, especially Claire Kanja for teaching me all the skills I needed and Prof. Kim Hammond-Kosack and the Fusarium lab group for their advice and encouragement.
IMAGE: Karen in the Centenary lab at Rothamsted Research (summer 2020)