Genome Insider S5 Episode 4: Gotta Catch ‘Em Gall

Menaka: So usually, we start an episode at a sample site — and we’ll get there, but today, let’s start at the very beginning — before there were any samples, or project proposals. Way back when this project really first started.

Kasey Markel: And, uh, it started because I am too willing to say yes.

Menaka: This is Kasey Markel, 

Kasey Markel: and I’m a PhD candidate with Patrick Shih working at the Joint BioEnergy Institute at Lawrence Berkeley National Lab. 

Menaka: And here’s Patrick Shih, 

Patrick Shih: I’m an assistant professor of plant and microbial biology at UC Berkeley. My group is very focused on how we can engineer plants. We love to be inspired by nature.

Menaka: And we’re talking about a conversation super early in Kasey’s PhD.

Kasey Markel: So I’d spent maybe like three months full time in the lab. And one of the postdocs at the time, who was kind of our first post doc and the one setting up the lab, told me that Patrick had just come downstairs and proposed a project to him that he had immediately said no to.

Menaka: Bit of a stalled start. But you know this project gets off the ground, because we’ve made an episode about it. Patrick just hadn’t pitched the idea to the right person yet.

Kasey Markel: And then a couple hours later, Patrick, which is my Ph. D. supervisor, came down and proposed a project to me. It began with one word. Wasps! 

Patrick Shih: <laughs> Yeah, I do remember. That was like the first few months of being a professor.

Menaka: Not just wasps, exactly — Patrick had stumbled on these weird structures that some wasps use to raise their young. They caught his eye because wasps don’t build these structures themselves — they hand the hard work off to a plant. 

Basically, these wasps lay their eggs in part of a plant. But then, the baby wasps don’t hatch and leave. They grow up inside the plant! Because these wasps are able to inject chemical instructions that tell a plant to grow a nice, protective pod around their baby. The specifics of this process, and what these instructions are made of is still pretty mysterious — but this works! 

These baby wasp pods come in many different forms. They can look like tiny grapes sitting on a leaf, or lumps in a plant’s stem, or spiky fruits along a branch. Basically, something that’s made of plant ingredients, but… different instructions. The wasps are engineering plants to grow extra structures. And they’re totally different from a plant’s normal roots, shoots, leaves and fruits. 

Patrick Shih: I was very humbled by how good nature could come up with an elegant solution to doing many of the things that we fail to do on a very daily basis in the lab.

Our lab is interested in plant synthetic biology and ostensibly, like, the field is very interested in, like, how could we control whatever facet of a plant? And we try really hard and fail. And here’s this great example of just, like, this tiny wasp that just, like, pokes its butt into a leaf. And like, makes it do whatever it wants. So that its babies can survive, it’s protected from the elements.

Menaka: So, Patrick had pitched Kasey a pretty wild thing to look into.

Kasey Markel: And I thought, “That seems interesting. I’ll do it as a side project.” Didn’t really think it would go anywhere, but turns out it went somewhere. 

Menaka: Well, that’s great. Saying yes to a project called “Wasps”, there’s probably worse things you could have done. 

Kasey Markel: Absolutely. I’ve made far worse choices in my PhD.

Menaka: So in this episode, we’ll hear from Kasey and Patrick about how this project unfolded, and how they worked with the JGI’s metabolomics program to find out more about these weird little pods. Or, to be more brief, WASPS. 

This is Genome Insider from the US Department of Energy Joint Genome Institute. Where researchers discover the expertise encoded in our environment — in the genomes of plants, fungi, bacteria, archaea, and environmental viruses — to power a more sustainable future. I’m Menaka Wilhelm. 

Today, a project to see what wasps can teach us about engineering trees. So let’s get back to that structure that Kasey signed on to study. This kind of growth is called a gall – like a ball, but with a g.

Kasey Markel: So, galls are abnormal structures on plants induced by other organisms. And they’re often bright colors. These particular galls, they look like little fruits that are growing out of the leaves of an oak tree.

Menaka: And in Kasey and Patrick’s work, they’re looking at structures that a specific kind of wasp — the Cynipid wasp — makes on oak trees. But it’s a big wide world out there.

Patrick Shih: You know, it’s not just wasps, there are other insects that will induce galls on different plants, not just oak. 

Menaka: Also the wild rose, some spruce trees, the lime tree, the list goes on. And you might have come across them.

Patrick Shih: Like they look totally like, something’s not right with this leaf. You definitely are just like caught off guard, like that’s not what a leaf looks like.

Menaka: For example, does that oak leaf have spotted yellowish grape growing on its underside? Or, why is that rhododendron growing a tiny, lumpy green apple along with its leaves?

Kasey Markel: Most people have observed some kind of weird thing on a plant and been like, “That’s not a fruit.”

Menaka: Nope! But depending on what you’re looking at, it could be a gall. Plus, there’s what’s inside the gall — a baby insect that the plant is basically providing full-service babysitting for. 

Patrick Shih: It’s a pretty nice bed and breakfast for those babies. You know, they just sit there and they’re like protected. They’re nice and cozy. They get sugar water fed to them. 

Menaka: And it’s pretty complicated to build a baby insect bed and breakfast, actually. We don’t know exactly how Cynipid wasps get plants to do this — but it’s clear they’re doing a lot.

Kasey Markel: They’re making the plants generate this de novo organ, according to the design specifications of the wasp, that it can live in and be fed by for its entire lifespan. 

Menaka: These wasps only live in the galls while they’re growing into adults — but some insects who build galls get all of the nutrition that they’ll need for their whole lives from these galls. The goldenrod gall fly is one of these insects. 

Kasey Markel: The fully adult form actually emerges from the gall without even having any teeth remaining or any mouth remaining. It’s incapable of eating, it just gets all its food during its development, then it just leaves as a fully fledged adult, mates, makes new galls and dies.

Menaka: Wow. 

Kasey Markel: So they’re used for both a house and a food supply. 

Menaka: And somehow, these wasps and insects set all of that up — they push the plant to create a repeatable structure that’s totally different from what it normally grows, and very functional. 

Kasey Markel: So it’s something that human synthetic biologists have been trying to do, you know, we can do a little bit of plant genetic engineering, improve one trait, you know, maybe change the color, change the flavor, improve the resistance to insects. But the wasps are doing something much more complicated than humans have ever done.

Menaka: So, given what’s going on in galls — if we could crack even part of how insects organize this system, there are lots of possibilities.

Kasey Markel: In principle, if you have the understanding of plant biology that the wasps have somehow instinctually, you should be able to make a plant, make anything that that plant is physiologically capable of making. Plants are incredibly diverse, like metabolically and biochemically. So they should be able to make an extremely large array of compounds, an extremely large array of sizes and shapes of structures like fruits. 

Patrick Shih: The applications are endless, if you really had that level of engineering of any plant. And so that’s a very lofty goal. But you know, I think our more realistic goal is just like, this is a really cool system and if we learned anything, I think it would better the entire plant field. And so that’s kind of how we approached it. When we wrote this proposal to JGI, it was more focused on like, look how cool this is. Even if we learned anything, it would be amazing. 

Menaka:  So that was in 2019. They submitted a proposal requesting sequencing and metabolomics data from the JGI. The plan was to submit samples from oak trees, and the galls that wasps make on their leaves. And overall, this project looked at a pretty big question — what do wasps use to make these galls form and grow?

Menaka: Somehow, wasps manage to send a tree very detailed instructions on how to build a gall. Those instructions probably come from a mix of molecules — but exactly how those molecules tell a tree what to do? That’s still pretty mysterious. 

As Patrick and Kasey set up their plans, they thought about a few different possibilities, for like, what exactly tells tree cells that they should follow a wasp’s directions to build a gall.  

Kasey Markel: There’s a handful of different hypotheses for like — what is the key signal here? 

Menaka: As they thought about what molecular mix makes that key signal, they actually thought about another organism — much smaller than a wasp. This is a bacteria, but it also engineers plants.

Patrick Shih: So there’s another system that almost all plant molecular biologists use, which is this bacterium called agrobacterium. 

Menaka: Agrobacterium is pretty different from the wasps we’re talking about – but it has something in common with our wasps.

Patrick Shih: And the cool thing about agrobacterium is it’s actually a plant pathogen. And it will actually form galls. 

Menaka: So, not so different after-gall. And unlike these Cynipid wasps, researchers do know how this bacteria engineers plants. Agrobacterium changes what plants are doing by putting its own DNA into a plant’s genome. 

Patrick Shih: So that’s like a nucleic acid based method of modifying a plant. 

Menaka: Agrobacterium is a case where the key signal, or the gall directions, come from a nucleic acid — DNA. Kasey and Patrick figured maybe that was an important commonality to pay attention to.

Kasey Markel: It could be that the galls induced by these wasps are similar to the galls induced by agrobacterium, where it’s a particular nucleic acid that’s causing the change to the plant. 

Menaka: The other thing that’s interesting about this specific possibility — is that we understand agrobacterium so well that it’s used really widely in plant biology. Researchers have figured out how to work with that bacteria, so they can use it to engineer some plants.

Patrick Shih: And that’s how we transform plants, we can genetically introduce foreign DNA into a plant genome.

Menaka: But agrobacterium doesn’t work universally — so, if wasps could teach us another way of engineering plants with nucleic acids, the field’s options could eventually open up quite a bit. 

So if wasps directed plants to make these galls with nucleic acids, it would be a neat finding. But that’s just one hypothesis. There’s also, an alternative one. 

Kasey Markel:  That it’s not a nucleic acid at all. It’s instead some series of small molecules or metabolites, 

Menaka: And to test that hypothesis, they’d want to measure those molecules — with the right expertise.

Patrick Shih: So, metabolomics seemed to make sense. That’s one of the reasons why like JGI makes perfect sense to work with on this, specifically Trent Northen’s group. 

Menaka: So they had a few ideas of what to be on the lookout for, and next they’d pick which galls to study. Even narrowing down to wasp galls on oaks, there were a bunch of options – and they considered quite a few.

Patrick Shih: There was a good part of, uh, Kasey’s PhD, we were just like, “gotta catch them all.” Kinda got that Pokemon mentality. So –

Menaka: Gotta catch them gall, I guess. 

Patrick Shih: There you go, that was too easy.

Menaka: So next, that gotta catch ‘em gall phase. That’s after a quick break about how the JGI supported this project. 

Allison: The JGI enabled this project through its support of DOE Bioenergy Research Centers. In addition to supporting users, the JGI sets aside time and capabilities for PIs working with these centers. 

All JGI users have access to the same capabilities – so researchers who are submitting proposals to, say, the New Investigator or Functional Genomics calls under our Community Science Program can access the same tools as the BRC PIs. In this case, Patrick Shih and Kasey Markel’s proposal leveraged both targeted and untargeted metabolomics analysis from Trent Northen’s Metabolomics group. But you don’t have to take it from us, here are Kasey and Patrick. 

Kasey Markel: JGI had not only like all the machinery to run a metabolomics pipeline, but also a handful of experts that I could have a lot of back and forth with to try to understand my data better.

And I definitely want to shout out to Ben Bowen who had many dozens of emails and Zoom calls with me trying to help me understand exactly what was going on with this data to make sure I could present it well for the paper. 

Patrick Shih: I think most people could get untargeted metabolomic data and just be overwhelmed with the amount of data that you get from that. But to be able to ground that in some sort of ground truth from the targeted metabolomics and that’s actually – Trent’s group’s has been working at the plant microbe interface,so they have a lot of molecules that are somewhat relevant to the things that we would be interested in.

So that was another reason why Trent’s group made a lot of sense to work with. Arguably, one of the most important facets of this paper is having Trent be that collaborator to enable all of that research.

Allison: You can find out more about submitting proposals to the JGI on our website. Head to joint geno-dot-me slash proposals. We’ve also got a link to our website waiting for you, wherever you’re listening to this episode — either in the episode description, or the show notes. 

Menaka: So — as a quick recap, Kasey Markel and Patrick Shih have set off on a project with the JGI to try to figure out how wasps program trees, specifically, their leaves, to house and feed wasp babies in these structures called galls. Which is fascinating at a basic level. It could also lead to new ways of engineering plants in the future. To get there, of course, they had to collect samples. Here’s Kasey Markel, again.

Kasey Markel: I started this project at UC Davis and Davis is very blessed in that we have one of the largest collections of oak trees in the world. There’s a section of the Arboretum that has over a hundred different species of oak trees. 

And so I just looked at all of these trees for about a year. I looked at every species I found and I recorded every gall that I found. 

Menaka: This is that ‘gotta catch ’em gall phase that Patrick mentioned — because Kasey looked high and low. 

Kasey Markel: So I didn’t only collect inside the Arboretum. I also collected along the whole Putah Creek riparian area across like several miles. It was actually part of my commute to work. I had to cross this creek by kayak.

Menaka: Wow! Your daily commute includes a kayak trip? 

Kasey Markel: It used to. I was living on a 40-acre ranch outside of town. It was in the next county over, in Solano. But, I walked to the edge of the ranch, went down to the river where I had my kayak locked, and then I kayaked across the river. I was crossing Putah Creek, which runs from Lake Berryessa to Sacramento, more or less. And then on the other side, I had my bike locked to a tree, and I biked to work.

And I ended up collecting a bunch of samples right across my creek crossing because I saw a bunch of these galls on the local trees there.

Menaka: Commitment, I tell you! So between that UC Davis Arboretum, and the kayak-route sampling, they had plenty of different galls to choose from. So they wanted to pick a couple that looked different, and ask questions across them, to see if that got them closer to understanding where they’d come from.

Patrick Shih: Are these galls really the same thing? They’re different colors and like slightly different shapes. Or are they like fundamentally different at a metabolite level, at a tissue level, at a broader morphological physiological level? 

Menaka: So Kasey sifted through the leaves he’d collected, and picked two types of galls. They come from the same group of wasps, but not exactly the same insect. And they stood out for being some of the most eye-catching.

Kasey Markel: One of them is purple and urchin shaped and kind of spiky. One of them is red, cone shaped, kind of like a wizard’s hat.

Menaka: Besides being brightly colored and weird-shaped, these galls had another thing going for them.

Kasey Markel: And they also both were generated from the leaves, and the tissue was relatively soft, parenchymous tissue, kind of. I compare it to, like, the texture of the inside of an apple, versus other types of galls that are more woody. 

Kasey Markel: And the reason why that’s very important is getting metabolites or DNA or RNA out of, like, wood is extremely difficult. I spent some time trying, it did not work, and these ones I thought were much more likely to be biochemically amenable to modern analysis techniques. 

Menaka: And so these were the two galls they looked at, to try to understand how they formed. Were the wasps using nucleic acids to convince trees to run their nurseries? Proteins? Small molecules? A mix? 

To get at that, they looked at ordinary leaves as a baseline, and watched what changed in leaves with galls. And they had samples from different stages of those galls forming. 

Kasey Markel: What’s really nice about that is we can track which molecules are spiking really early. So obviously anything that spikes really early is a candidate for something that is required for the induction of the gall as, or potentially something that the mother wasp put there.

Menaka: And there’s more than one molecule that causes these galls, that’s clear — but after these experiments, and then others from other teams, the wasps don’t seem to be using nucleic acids to get these galls built.

Patrick Shih: The jury is still out, but we’re pretty confident that’s not the mechanism. 

Menaka: Instead, it seems like wasps use a mix of small molecules, like plant hormones, and proteins, to get trees to create these structures.

Kasey Markel: We know a handful of the key molecules involved in that process. We know that they manipulate the hormonal structure of the plant leaf, they change the plant hormonal profiles. 

Menaka: Initially, they hoped to connect these molecules to the genes that turned on when galls formed, with transcriptomics. That didn’t end up working out,

Kasey Markel: Turns out oak RNA is very difficult to work with. 

Menaka: So it’s too soon to pinpoint an exact mix of stuff that makes a gall — but they did find something pretty wild about how these galls siphon food and water toward the baby insects they house. This has to do with the circulatory system, the vasculature between these galls and their trees. 

Patrick Shih: And so if the purpose is to feed these larvae, the vasculature is like the plumbing of the plant. You can send the water, you can send food over to wherever you want. 

Menaka: And researchers have known for a while that galls are fully tapped into a tree’s channels for water and food, which is super important. What Kasey found out, is that within a gall, its circulatory system develops from scratch, totally separate from the leaf it’s sitting on. Then, later, it connects into the plant’s existing plumbing.

Kasey Markel: We actually showed de novo vascular systems that were developing within the gall itself and then later connecting to the plant vascular systems. And one of the cool characteristics of that is that the gall vasculature and the plant vasculature look kind of similar but are not exactly the same.

Menaka: Because basically, a wasp directs the plumbing construction of one system, where normally, a tree runs its own plumbing. Patrick had a great analogy for this — if you think about the tree as a city, and the gall as a new park, 

Patrick Shih: It’s non-trivial to just be like, Hey, I just went to this park and I want plumbing to come here. You go through the city. You gotta like, dig up holes and everything like that. 

Menaka: Quite a project! 

Patrick Shih: And so. To all of a sudden be like, “No, no, no, like, this is where I want this gall to be, and I want you to feed my babies,” we still are baffled by how this actually happens. De novo vascularization is still a real head scratcher, but if we can figure that out, that would be super cool. 

Menaka: Both Kasey and Patrick have plenty of other ideas about what this work might lead to in the future.

Kasey Markel: One of the most interesting future lines of research is taking some of those molecules that I identified as highly prevalent during the early stages of gall development and injecting those either singly or in combinations into the leaves of oak trees and see if you can generate de novo galls.

And again, the moonshot is if you can recapitulate the gall phenotype, and you know exactly which molecules are required there, then you basically have decoded what it is exactly that the wasp is doing. And once you have that basic recipe, you could do variations on that recipe to try to be like, well, does the gall morphology change? Does the gall get bigger or turn a different color or things like this? And eventually you can have this tunable, like, plant programming set.

Menaka: Which would be really pretty neat. There’s a long way to go before that’s in reach, but for Patrick, that’s kind of the exciting part. 

Patrick Shih: The major thing that I like to highlight in this story, why I get really jazzed about this work, is highlighting how little we know. In biology, molecular biology, we tend to like to gravitate around model systems because we can have this depth of understanding that you couldn’t have in a non-model system.

But that doesn’t mean that these other, non traditional, non model systems aren’t full of super interesting biology. And they’re very intractable. Like Kasey had to go hunting for these galls, there’s a field aspect to this research. But yeah, the most exciting thing to me is how remarkable this transformation is that we observe, but we know nothing about how this happens. And so like any Incremental, like, improvement in that understanding is humongous, 

That’s the thing I like to impress upon people, is just how little we know about the system. Despite many people being very interested for millennia about it.

Menaka: To wrap things up, Kasey pointed out a pretty notable person who’s been interested in these galls, too — one of biology’s heavyweights.

Kasey Markel: I think the story that we have today is slightly more detailed, but not that much more detailed than the description that was given at the end of the final chapter of Origin of Species.

Menaka: That’s right, we’re shouting out Charles Darwin, so here’s that quote, slightly shortened: 

Kasey Markel: Nevertheless, all living things have much in common. We see this even in so trifling a fact as the same poison often similarly affects plants and animals, or that the poison secreted by the gall fly produces monstrous growths on the wild rose or oak tree.

Menaka: In this case, the focus wasn’t how these galls form, specifically – but these weird little structures did show something important. 

Kasey Markel: Charles Darwin was using that as a claim for the common descent between very diverse types of plants, for his claim of common descent of all species. 

Menaka: As with many things, evolution connects them. And as they’ve advanced, these wasps have managed to accomplish something pretty amazing. As usual, the biology and engineering that we find in nature has a ton to teach us. 

So again, that was Kasey Markel and Patrick Shih from UC Berkeley and the Joint BioEnergy Institute, a DOE Bioenergy Research Center managed by Lawrence Berkeley National Lab.

We’ll link to their work in our episode description. You can also find a transcript of the episode there!

As always, you can learn more about how to work with us at jointgeno.me/proposals.

This episode was written, produced and hosted by me, Menaka Wilhelm. 

I had production help from Allison Joy, Massie Ballon, and Graham Rutherford. 

You heard music in the middle of this episode by Cliff Bueno de Mesquita, who used to be a postdoc at the JGI.

If you liked this episode, subscribe or follow wherever you’re listening, and help someone else find it! Tell them about it, email them a link, or leave us a review wherever you’re listening to the show. 

Genome Insider is a production of the Joint Genome Institute, a user facility of the US Department of Energy Office of Science located at Lawrence Berkeley National Lab in Berkeley, California.

Thanks for tuning in – until next time!