Over summer, I spent 8 weeks in the Warwick Integrative Synthetic Biology Centre, working in the lab of Prof. Vardis Ntoukakis. My project was built on a screen of more than 150 effectors from the oomycete pathogen Hyaloperonospora arabidopsidis, looking for an effector that could increase plant tolerance to heat shock without impacting other phenotypes such as immunity. If successfully identified and characterised, upscaled incorporation of such effectors could provide a valuable mechanism to reprogramme plants to mitigate the damaging impacts of heat stress on crop growth, thereby significantly increasing the ability for crops to grow in warmer climates; a factor that in a world of continuously rising temperatures could prove essential in food security and provisions globally.
I have been working with an effector identified from this screen, 6E6 with the aim to validate expression of 6E6 in both estradiol-inducible and 35S Cauliflower Mosaic Virus promoter 6E6 transgenic Arabidopsis thaliana lines. I also aimed to localise 6E6 via transient expression of green fluorescent protein (GFP) tagged 6E6 constructs in Nicotiana benthamiana.
To validate 6E6 expression, I grew the 6E6 transgenic Arabidopsis thaliana plants to seedling stage, and then treated them with estradiol for 24 hours. To detect 6E6 protein, I used immunoblotting with an anti-HA antibody. This enabled identification of 6E6 protein in the 35S promoter line and indicated possible presence of 6E6 in the estradiol-inducible line. Considering that 6E6 expression in the estradiol-inducible line may be lower, I performed a qPCR. This enabled me to confirm 6E6 expression in both lines, with expression levels 25-fold greater in the 35S promoter 6E6 line compared to the estradiol-inducible 6E6 line.
My second task to localise 6E6 involved creating 35S::6E6-GFP and 35S::GFP-6E6 constructs using Golden Gate cloning standards. I transformed these into Agrobacterium tumefaciens and infiltrated the constructs into N. benthamiana leaves, then visualised the constructs with confocal microscopy. This allowed me to see that the 35S::6E6-GFP construct was localising in a dynamic manner within the cytosol – future work involving co-localisation could help to determine more specifically whether localisation is taking place in the ER, cytoskeleton or another subcellular structure.
As an overall experience, I thoroughly enjoyed my time in the lab, particularly through the many opportunities to practice a wide range of laboratory skills spanning molecular biology, working with plants, microscopy, RNA and protein work – key techniques which will be invaluable to take forwards into the final stages of my degree and beyond. My confidence in the lab has grown considerably under the generous and extremely knowledgeable day-to-day guidance of Dr Ana Dominguez-Ferreras, and the overarching steer of my PI Vardis Ntoukakis. I am extremely grateful to the BSPP for their generous support that has enabled me to spend my summer gaining practical laboratory experience – an area that previously presented very few opportunities throughout my undergraduate studies as a result of the pandemic.
Emma Reilly
University of Warwick
Confocal microscopy of 35S::6E6-GFP by agroinfiltration in Nicotiana benthamiana showed the effector construct was forming dynamic structures in the cytosol.
