One day, a few years ago, a local teacher found something strange in his garden. The story that follows is one of plants, beer, bacteria, insects and ZOMBIES! For this Halloween special edition blog Dr Sam Mugford, research assistant, reveals the strange science of phytoplasmas, a group of plant-infecting bacteria with very peculiar properties, and how he made his own delicious zombie beer…

“In 2018 I saw a post on social media from a local teacher, who had spotted something strange in his neighbourhood.

“His hop plants were growing leaves from where the flower cones should be. As a plant scientist and entomologist, this piqued my interest. Firstly, I was interested because I like brewing (and drinking) beer. Secondly, I had my suspicions about the cause of this abnormal growth.

“In the Hogenhout lab at the John Innes Centre our team studies the interactions of insects with plants, in particular sap-sucking insects such as aphids, leafhoppers and spittle bugs.

“What these insects all have in common is specialised mouthparts, called stylets. These piercing, sucking organs penetrate the plant and extract the sap from the plant ‘veins’, like vampires. It makes these insects exceptionally good vectors for diseases, like viruses and bacterial plant pathogens.”

Bacterial plant pathogens that turn plants into zombies

“One of the plant bacterial pathogens that we study are called phytoplasmas. These are intracellular bacteria, which makes them difficult to study, because they only grow inside plant or animal cells. We can’t grow them in the lab on a petri dish, so we study them inside the plant, or indeed inside the insect.

“Phytoplasmas do very weird things to plants. They don’t just infect them and make them sick. They take control of the plant’s development, often altering flowers to create leafy structures in the place of petals. Essentially, the bacteria turns plants into zombies, hijacking their development for their own benefit.

“You can see the effects of phytoplasma in all sorts of plants, including crops. This includes chickpea plants, the Madagascan periwinkle (see the image gallery below), Arabidopsis, and oilseed rape. In fact, phytoplasmas can be a significant pest to oilseed rape crops in many areas of the world. Aster yellows is a common name for the disease.

“These effects are very similar to what I saw in the social media post of the hops, where instead of the classic flower cone structure, there were leaves growing instead. My first thought was that this could be caused by a phytoplasma.

“A pine tree which has a phytoplasma infection will grow branches wildly, producing an out-of-control bushy structure. With another nod to Halloween, these symptoms are known as witch’s broom. As well as leafy flowers and the bushy structures, there’s another change to floral developments that the phytoplasmas induce – indeterminacy.

“Generally, flowers are produced according to a specific plan, and when they grow they form petals, sepals, anthers, stamens etc, and then they stop. However, phytoplasma-infected flowers become indeterminate, and they continue growing leafy flowers out of the middle of the floral structures. The phytoplasma has taken over control of the plant development.”

But why? What benefit does this have for the bacteria?

“It turns out that some of the symptoms that are induced by phytoplasmas are attractive to the insect vectors. These insects are not pollinators. They are not interested in flowers, and they don’t like bright, gaudy colours. They don’t want pollen, they want sap, so they’re interested in nice, green, bushy plants. This is exactly the type of plant that the phytoplasmas cause.

“When you do experiments giving leafhopper insects a choice of an infected plant or an uninfected plant, they are more likely to pick the infected one and will lay their eggs there, or feed from it, which is how they pick up the phytoplasmas. The changes to plants caused by the phytoplasmas are actually helping the bacteria’s life cycle, because they’re encouraging the insects to pick up and spread them.”

Why do we think of infected plants as zombie plants?

“Plants that are phytoplasma-infected are no longer able to reproduce. The flowers are sterile, they can’t produce seed. They live longer, their life cycle is extended. They live only to serve their bacterial masters.

“The idea of zombies in nature is not unique to phytoplasmas, as there are plenty of examples where the infection of organisms by parasites resembles this zombie-master relationship. Snails can become infested with fluke worm, which causes a green and bulbous growth of its antennae, which pulsates and mimics caterpillar movement to attract birds to feed (see image gallery below). The worm does this as caterpillars are particularly attractive to birds, so this change encourages the birds to come and eat the snail and thereby spread the infection. Again, the parasite is taking control of its host for its own benefit.

“Another example in plants is a fungal pathogen that infects a plant called a Bochera, in the cabbage family. In almost the opposite way of phytoplasmas, it causes the growth of leaves that resemble flowers. The fungus produces spores from the floral-like structures. These structures attract pollinators which, instead of picking up pollen, accidentally pick up fungal spores and spread them.

“Similarly, cordyceps is a group of fungi that infect insects, most famously ants. A cordyceps infection changes the behaviour of ants, causing them to climb up to the tops of trees, and this is when the fungus produces its fruiting body. This mushroom, which grows out of the ant’s head, distributes fungal spores high into the air.

“Cordyceps are perhaps more famous than they used to be, rising to fame as the supposed cause of the zombie apocalypse in the game, ‘The Last of Us’.”

How do phytoplasmas control the development of the plant?

“This has been the focus of work in Saskia’s lab for many years, and we have found that once inside it, the phytoplasmas secrete a number of proteins into the host plant cell.

“The research group has identified more than 50 different proteins that these bacteria can secrete into the plant cell. Here they interact with cellular processes in various ways. We investigate what those proteins do, and how they affect plant development.

“One way that we do this is to identify the genes from the phytoplasmas that encode for the proteins, and we introduced these into plants so their cells produces just one of these proteins. Then we investigate how they affect the development of the plant.

“We asked questions like, “Do any of the proteins cause leafy flowers? Branching? Or, make plants more attractive to the leafhoppers?” And to cut a very long story short, yes they do.

“So far, we have identified and worked on three proteins: SAP11, SAP05, and SAP54. SAP11 and SAP05 both caused our test Arabidopsis plants to have increased branching, resembling phytoplasma-infected plants. SAP54 caused the flowers to turn into the leafy structures.

“Remember, this is not in the presence of an infection by phytoplasmas. This is just the action of one protein alone. We’ve also found with all three of these proteins, when you make plants express them the plants become more attractive to leafhoppers.”

What has that got to do with hops and beer brewing?

“As soon as I saw the hops I thought, “You might have a phytoplasma in there.” I got in touch with the person who posted, and they happened to live down the road, so he came to the John Innes Centre in Norwich and brought some of his potentially phytoplasma-infected hop flowers, as well as some asymptomatic flowers with him. I extracted DNA and did PCR tests to try and detect phytoplasma, and in the symptomatic flower we could detect DNA from a phytoplasma.

“For me, the obvious next step was to make some beer out of the infected hops. So, we took the infected hop flowers into my beer brewing shed at home, and then wondered what sort of malt we could use for this special “zombie” beer.

“Coincidentally, a few years ago a colleague called Dr Chris Ridout found a heritage barley variety called Chevallier in the Germplasm Resource Unit, which had really useful malting and disease-resistant properties. He resurrected this variety and it is now grown and used commercially.

“What better than using a malt barley that was brought back from the dead for a zombie beer? Using the Chevallier malt, and the zombie hops, we made a delicious zombie stout.

“My advice — keep an eye out for your own zombie plants this spooky season. But don’t worry, these pathogens aren’t dangerous to humans or animals.”

 

With thanks to: Ben Dewhurst, head of science at Wymondham College, Lawrence Percival-Alywn, Research scientist at NIAB, Cambridge, and associated scientists Akiko Sugio, Allyson Maclean, Zigmunds Orlovskis, Pascal Pecher, Weijie Huang, Qun Liu, and Saskia Hogenhout.