Unleash your inner Ninja — when the research tempo accelerates
Scientific research advances at different paces depending on the project. But sometimes, you may want to unleash your inner Ninja and fully press on the gas pedal.
Cite as: Kamoun, S. (2022). Unleash your inner Ninja — when the research tempo accelerates. https://doi.org/10.5281/zenodo.7143548
Genetic analysis in potato — the third most important food crop — can be painstakingly slow. To identify a potato gene using positional or map-based cloning is not just slow, but uncertain. One needs to identify the appropriate genetic material from diverse, often genetically heterogeneous populations, make the appropriate crosses, hope that the phenotype segregates as a Mendelian trait, and that the genetic mapping work goes smoothly. Even after zooming in on a defined genetic locus, validation of the candidate gene may fail, leaving the scientists with little to show after years of work. The whole process requires patience and persistence.
I don’t count patience as a virtue. Leo Tolstoy wouldn’t think much of how I live my life given that he wrote, “the two most powerful warriors are patience and time.” I do listen to Yoda’s, “patience, you must learn.” But my favorite science projects are the ones that cycle rapidly, when we can quickly test, and generally discard, numerous hypotheses. This is why I love the model plant benthi — Nicotiana benthamiana — which allows just that. Fast-forward plant biology, we like to call it. Weekly cycles of iterative experimentation that accelerate hypothesis testing and allow us to rapidly plow ahead in solving biological puzzles.
My impatient nature is why I have huge respect for my colleagues who undertake map-based cloning in challenging but important experimental systems like potato and its relatives. Take for example this recent paper by my friend and long-term collaborator Vivianne Vleeshouwers. Vivianne and her Wageningen University & Research Plant Breeding colleague, Jaap Wolters (aka Pieter J. Wolters), set up disease screens of wild potatoes in 2014. One year later, they discovered accessions resistant to the fungus Alternaria solani, the causal agent of early potato blight. In 2016, they obtained seeds from the first crosses with a resistant genotype of the wild species Solanum commersonii. They could map the resistance gene to a 3 Mb region at the top of chromosome 12, but to move forward they still needed to sequence the genome of the resistant plants and fine-map the locus. For this, they screened 3,000 (yes, three thousand) plants from the cross to reduce the interval to ~20 kb. This allowed them to finally identify the two genes that confer fungal resistance. It turned out that these genes code for glycoalkaloid metabolites, which act on various fungi but also on the insect pest the colorado potato beetle. And on September 30, 2022, they published the paper, a full 8 years after the first experiments. Phew!
Map-based cloning is a tried and tested method. Back in the 1990s, plant biologist Abdelhafid Bendahmane, working at the time in my current home institute The Sainsbury Laboratory (TSL) with Konstantin (Kostya) Kanyuka and David Baulcombe, identified the virus resistance gene Rx from the Andean potato Solanum tuberosum ssp. andigena. Abdel’s trick was to pull out the gene from a BAC (Bacterial Artifical Chromosome) library and rapidly validate it using a number of plant transformation methods. This was the first potato gene to be cloned by the map-based method. At the time, it felt to me like a heroic feat.
Abdel went on to have a fabulous career at INRAe, cloning, among other things, several agronomically important genes from crops like melon, cucumber, tomato, and pea. Recently, INRAe recognized Abdel with a Gold Medal (médaille d’or). And Rx, the resistance gene Abdel, Kostya and David cloned almost a quarter of a century ago, is still being studied by TSL PhD student Mauricio (Mau) Contreras. Mau recently produced our most advanced mechanistic model of how Rx activates immunity. More on this later.
Also here at The Sainsbury Laboratory (TSL), my next-door colleague Jonathan Jones (JJ) has completed dozens of positional cloning projects throughout his rich career, both in potato and other plants. Over the years, JJ and colleagues came up with various tricks to accelerate the cloning process. But even when the molecular biology can be streamlined, there is still the need to screen germplasm for interesting phenotypes, as well as the tedious process of performing and analyzing the genetic crosses.
Xiao Lin, a postdoc in JJ’s lab, is a marathon runner, as you might expect for a scientist engaged in potato research. Recently, Xiao has further accelerated the process by combining genomics with pathogen (virulence) effector screens — aka effectoromics — to efficiently identify three new late blight resistance genes from the wild species Solanum americanum. His work is truly spectacular. But as Xiao’s earlier work with Vivianne — who, incidentally, pioneered these effector screens about 15 your ears ago — illustrates, map-based cloning can be a cruel exercise. In one project, the last step — the validation of the candidate genes — turned out to be problematic. Xiao candidly reported the heartbreaking results in his thesis summary:
“Attempts were made to perform complementation tests of these candidate G-LecRK genes by co-expression with the matching SCR74-b3b effector, however, I have not been able to confirm the identification of the SCR74-B3b receptor yet.”
All this painstaking map-based cloning of disease resistance genes from wild relatives of crop plants is incredibly valuable for agriculture, justifying the time, effort and cost that Vivianne, Jaap, Abdel, JJ, Xiao and others invest in this type of research. The genes not only help guide plant breeding for disease resistance but can also be combined through transformation in a single potato plant to confer robust and hopefully durable resistance to the pathogens. Check for instance the 3R Victoria potato being trialled in East Africa for providing resistance to the Irish potato famine pathogen Phytophthora infestans. Thanks to the combination of three cloned resistance genes, African farmers can grow this potato variety without relying on fungicides. This is no small feat. In countries like Uganda, potatoes are grown not just for income but also as a subsistence crop. And because of chronic misuse and lack of personal protective equipment (PPE), fungicides can directly affect farmer’s health.
This is all to say that map-based cloning and similar types of research are comparatively slow paced, but for very good reasons. However, occasionally, opportunities arise that require a different approach, a fast-paced approach. We jokingly call such research projects Ninja projects. And these Ninja projects became the stuff of legend in our lab.
One such Ninja project dates back to early 2013, when my colleague Vladimir (Vlad) Nekrasov, currently at Rothamsted Research, and I made the executive decision to ditch the other projects we were pursuing at the time to focus on testing the CRISPR/Cas9 gene editing method in plants. Things went really fast from there. It took Vlad just a couple of months to get CRISPR to work in benthi. By mid-March, he had the first sequence validation of the edited genes. A few weeks later, we wrote the paper and by August it was published.
Although Vlad pretty much single handedly led the CRISPR/Cas9 project, most Ninja projects bring together several lab members who join forces to complete the work at an exceptionally rapid pace. This is another reason why I love Ninja projects. They foster cooperation and team spirit. The famed esprit de corp — the feelings of pride and mutual support — that is so critical to the success of any team. TEAM — Together. Everyone. Achieves. More. By joining forces on an intense, focused, short term project, the team gets the project completed in record time. And then, as the saying goes, a rising tide lifts all boats.
This exactly happened with the so-called MADA project of 2019. The project kicked-off after Jizong Wang, Jianmin Zhou, Jijie Chai and colleagues reported the first structure of a plant resistosome, the immune receptor protein complex that confers resistance to pathogens. At that point, we had an Aha moment, and realized that we could make sense of some old data we had. As I wrote in an earlier article:
“When the paper of Jizong Wang and colleagues describing the resistosomecame out in early 2019, we got very excited, because we had some data in the lab suggesting that the N terminus of an NRC is really important for its activities. About a year and a half before that, Hiroaki (Aki) Adachi, who was a postdoc in the lab at the time, performed a transposon mutagenesis screen — a truncation screen, if you like — of NRC4 to find out if we could get shorter versions of this protein that are autoactivated. What Aki discovered is that the very N terminus of NRC4, its first 29 amino acids, are sufficient to trigger the cell death hypersensitive immune response. To be honest, we really didn’t know how to interpret this result. We knew there was something important there, but we just didn’t know how to make sense of it. So it went in the drawer of intriguing results and Aki moved on to projects with better prospects. This is why when we learned about the death switch of ZAR1 and the funnel-like structure of the ZAR1 resistosome, it was an Aha moment for us. Right there we had a clear explanation of the functional importance of the N terminus, and to top it up a mechanism to explain how it would cause cell death.”
That’s when we made the executive decision to drop everything else and go Ninja Warrior mode. Aki called it the Ninja motif, and with Mau and the rest of the Ninja team, they wrapped up the paper and published a preprint in July 2019. It was just a few days before we presented the work at the Congress of the International Society for Molecular Plant-Microbe Interactions in Glasgow. It was a gratifying time for the Ninja Team as they worked so hard to advance the project at a dizzying pace. A MADA, MADA, MADA, MADA World!
Incidentally, a key finding of the MADA project was that a good number of NLR immune proteins like Rx — the disease resistance gene that Abdel, Kostya and David Baulcombe cloned almost 25 years ago — have lost their N-terminal MADA motif throughout evolutionary time and rely on so-called helper NLRs to execute the immune response. More recently, Mau Contreras along with Hee-Kyung Ahn in JJ’s lab also went full Ninja style to capitalize on the technical breakthrough of visualizing NLR resistosomes using non-denaturing gel electrophoresis. Although their first conclusive experiments were obtained in August 2021, they managed to complete the studies and publish their respective papers in April 2022 (find them here and here). It’s useful to ponder how our understanding of Rx and related NLR receptors has advanced over a quarter century in accelerated bursts of research breakthroughs, generally driven by methodological or technical advances.
The MADA and Rx projects both involved close coordination with the lab of our Imperial College collaborator Tolga Bozkurt who contributed their expertise in plant cell biology and biochemistry. That’s how Ninja projects work too. They can also fuel collaborations between labs, further expanding the research network and capitalizing on each team’s expertise. It’s precisely to foster such collaborations that we organize SchoBozKa, an annual joint lab meeting of the (Sebastian) Schornack, Bozkurt and Kamoun labs. This last year, when we met at Clare College in Cambridge, the event morphed into SchoBozKaDeRella bringing in the newly minted labs of Lida Derevnina and Phil Carella.
Tolga, his collaborator Doryen Bubeck and their team members, notably PhD student Tarhan Ibrahim, recently published a fantastic paper that’s also the result of a speed up Ninja style project. The project builds on game-changing advances in structural biology brought upon by AlphaFold, the AI system developed by DeepMind to predict protein structures from sequence alone. What Tolga, Doryen, Tarhan and colleagues discovered is that AlphaFold-Multimer — a variant of AlphaFold that predicts protein complexes — performs quite well in predicting the short linear motifs that the autophagy modulating protein ATG8 binds to. This is a massive advance because predictions of the ATG8 binding motif (known as AIM or LIR) based on sequence alone tend to yield too many false positive given that the motif is short and degenerate.
The computational pipeline developed by Tarhan Ibrahim et al. is incredibly impactful given that the cellular process of autophagy — the removal of unwanted molecules and organelles — modulates health and disease in humans, plants and all other eukaryotic organisms. Discovering a new pipeline to fish out autophagy associated proteins is exactly the type of science that requires dropping everything else and wrapping up the project in record time to get the knolwedge out to the community and accelerate the research. The authors clearly articulated the impact of their work when they wrote:
“We conclude that the AF2-guided discovery of autophagy adaptors/receptors will substantially accelerate our understanding of the molecular basis of autophagy in all biological kingdoms.”
Ninja-tai — Learn to collaborate
Ninja often work in groups like the Science Ninja Team Gatchaman (科学忍者隊ガッチャマン). This Ninja team or Ninja-tai focuses on environmentalism and responsible use of technology. I say it again, team work is so important. I recently visited with colleagues in industry. The question of what they look for when they hire academics came up more than once. The answer was simple, the capacity to collaborate is absolutely non-negotiable. And more often than not, academia doesn’t prepare well for this. Our perverse emphasis on individualism, silly metrics, and incentives against collaboration don’t prepare for a professional world where collaboration is sine qua non. As I wrote before:
“[The lack of collaboration] is one of the reasons why academia seems so cold to budding scientists. Many feel that the incentives of academic life don’t foster team work and the collaborative spirit that is central to our well-being.”
Unleash your inner Ninja, sometimes
Most research projects are like running marathons, they require stamina and are long haul efforts. But now and then an opportunity may arise to bring your team and collaborators in a huddle and put on your Ninja costumes. Go get ’em, Ninja-tai!
I thank Saskia Hogenhout for comments on an early draft. I’m also grateful to all the Ninja Teams I have worked with over the years, many of whom I didn’t mention here.