In conversation with Dr. Alisdair Fernie

Abstract

In this interview, Dr. Fernie reflects on his journey into plant biology—a path initially dominated by mammalian biochemistry but forever altered by a set of inspirational lectures and a timely opportunity at Oxford. He shares insights into the development of cutting-edge techniques to study protein–protein interactions (PPIs), the scientific motivations behind his publication ‘Investigating the dynamics of protein–protein interactions in plants’ which was awarded TPJ's Outstanding Technical Advance Prize, and the broader goals of his laboratory in decoding metabolic function and its genetic regulation. Beyond science, he opens up about the challenges of maintaining work–life balance, the joy of mentorship, and the importance of loving what you do.

1. Can you tell us about you, your childhood, and your educational background? Anything that you're comfortable sharing.

I was born just outside of Cambridge and lived near Leicester and in Hong Kong before settling in Maidenhead, a small town west of London. In secondary school, I developed early interests in various scientific disciplines and geography, alongside a passion for running.

2. How did you become interested in plant biology? Were you into plants growing up or DID that COME later in life?

My interest in plant biology came later. Having studied and greatly enjoyed Biology, Chemistry, and Geography as A-levels, I chose to stick with two of these and study Biochemistry at the University of Sheffield. The course I took was very much dominated by mammalian research, but I quickly realized that many of the practical studies were not for me and ended up doing a computational undergraduate project in protein structural biology in the laboratory of Dr. Peter Artymiuk, which yielded my first ever publication (Hempstead et al., 1997). Around this time, I also had my first plant lectures at Sheffield with Drs. Neil Hunter and Prof. Paul Horton, both of whom were very inspirational teachers and had a profound influence on my choice to switch to plants. When the opportunity to do a Ph.D. in Metabolic Regulation at the Department of Plant Sciences at the University of Oxford arose, my path was set. As I have discussed elsewhere (Fernie, 2014), on leaving Nick Kruger's Lab following my studies, the predominant advice I had was to change subject dramatically and take a post-doc in developmental biology or genetics. I ignored this advice and headed off to the Max Planck Institute of Molecular Plant Physiology to work with Lothar Willmitzer in 1999. In a relatively short time, I was given my own group and remained there to this day.

3. Would you summarize the main problem you and your team are tackling in this paper? What are the main differences between the different methods (BiFC, FRET, BiFC-FRET, BRET) you used to investigate PPIs? What are the advantages of one vs the others?

All the methods are for in vivo testing of PPIs in plants. BiFC is ideal for static visualization of protein interactions but lacks real-time dynamics. They all have advantages and disadvantages; however, to increase accuracy when reporting, using multiple methods to document the interaction is a standard requirement for publishing. To briefly compare and contrast the methods, FRET provides real-time interaction data but requires close proximity between proteins and careful fluorophore selection, BiFC-FRET offers both spatial and temporal information as well as the identification of three protein interactions, but is more complex and still suffers from potential fusion protein interference, and BRET allows for real-time interactions with less background noise and is great for high-throughput assays but requires the use of a luciferase substrate with low signal information in plants. With the suite of vectors we offer, these methods can be applied depending on whether you are interested in observing static or dynamic interactions, and whether you need to monitor interactions in living plants or in model organisms, such as Arabidopsis.

4. What are the main findings and contributions of this paper to the field?

The paper describes a resource set for PPI identification and validation. We had been carrying out our studies of enzyme–enzyme interactions of the Tricarboxylic Acid (TCA) cycle (Zhang et al., 2017) and glycolysis (Zhang et al., 2020). Whilst in these studies, we had already developed some of the vectors described in Zhang et al. (2023), many are described here. We reasoned that such a vector suite would be highly useful to many groups, and if we made this freely available would prevent them having to ‘reinvent the wheel’. Above and beyond the technical aspects, the development of a BiFC-FRET allowed us to establish that the glycolytic metabolon was responsive to substrate supply since the addition of glucose but not its poorly metabolized analog 2-deoxyglucose resulted in an increase in the glycolytic enzyme–enzyme complex.

5. What was the most challenging experiment in this paper?

On this one, I had to consult with the first author, Youjun Zhang, who told me the following: ‘Given that the labeling of the Halotag and provision of substrate to nano luciferase decreases the efficiency of energy transfer, the efficiency of nano BRET is very low in plant systems. This, unfortunately, means it cannot currently be used for analysis of dynamics. Moreover, given that both split luciferase and BRET could be used to test the protein interaction, we tried to use split-luciferase-BRET to test the dynamics of three or four protein complex formation in vivo. However, unfortunately, the signal is very weak. It is possible that the addition of helper peptides may improve the efficiency of this technique’. However, these need to be identified first!

6. In your opinion, what are the major challenges in your field? How does your overarching research tackle these challenges?

I think I must answer this twice, once concerning the work we have been discussing up until now, and the other time to cover the wider research being conducted in my laboratory.

In the first case, I believe we have successfully answered the ‘what question’ or, in this case, which enzymes form dynamic functional enzyme–enzyme assemblies and which mediate substrate channeling, but we lack resolution as to how these assemblies form and why? These questions need answering.

Outside of spatial understanding of metabolism, my laboratory generally studies its genetic control. We do so by evaluating differential metabolite accumulation in breeding populations and association mapping panels. It has become incredibly easy to elucidate which genes should be altered to improve the accumulation of virtually any metabolite. Twenty years ago, I would not have believed how facile this has become. However, what is currently limiting is information concerning the (precise) function of metabolites. This is urgently needed if we are going to fully exploit our capacities and breed more robust and nutritious plants as well as maximize their medicinal properties.

7. How about work–life balance? How do you manage all the work necessary to be both a successful scientist and a functional person?

The honest answer is that this is very difficult. The demands on scientists are quite varied and numerous, such that keeping on top of them, at least for small periods, has become key. Grant and paper writing are essential, but so is supervision. Many scientists have a much greater teaching load than I; however, I am highly active as editor of several journals (including my role as senior editor at The Plant Journal), and often review multiple papers per week.

I think the key to work–life balance is discipline and good time management, but also fully focusing on one thing at a time. In this way, I manage to juggle my work, my family (we have a 9-year-old daughter), and my hobbies (running and reading novels). I additionally travel much less than I used to, as the pandemic illustrated to me that travel is not as necessary as I had previously thought.

8. What is the most rewarding part of your job?

This has changed over time. As a young PI, I would have answered that having my research recognized in the form of a paper published in a high-caliber journal or a big grant funded, but now I would say it is definitely when someone in the laboratory gets a good position and develops into a strong independent scientist. The first author of the paper you highlight—Youjun Zhang—is a good example; he was in the laboratory for over 11 years, first as a PhD student, then as a post-doc, and finally as a project leader, but 18 months ago, he left to take up a position as group leader in the Institute of Genetic and Developmental Biology, Chinese Academy of Sciences, Beijing, where he is currently excelling.

I also greatly enjoy collaborating. I have amassed a massive network of collaborators worldwide, and doing science in collaboration either with scientists who share my expertise or those with entirely different skill sets is very fulfilling.

9. How about the things you dislike about it?

I am not fully happy with the fact that most funding is so short term, as this is a massive obstacle to visionary research. I also find it too high-pressure, especially for early-career researchers.

10. What advice would you give to young scientists who are starting their careers in plant biology

I was advised many times when I was finishing my PhD to change to a different field of research within plant science. Fortunately, I ignored this advice. I think the most important thing is to love what you do, so if you find something you like, stick with it. The same holds with collaborators—work with people you have an affinity with. And finally, don´t be afraid to ask for advice—people are (almost) universally happy to give it, and there is certainly wisdom in crowds!

11. If you were a plant, what would you be?

This is a tricky question. After trying to define my characteristics (and then find a plant that shared them) for several minutes, I gave up. My favorite plant is the weeping willow (Salix babylonica); there were several of these growing on an island close to my childhood home in Maidenhead, so I will be that.