Danish researchers trying to make crops more resistant to plant pathogens

Environment and sustainability 26. mar 2020 8 min Professor Hans Thordal-Christensen Written by Kristian Sjøgren

Researchers from the University of Copenhagen will develop a new method of making crops such as barley and wheat more resistant to plant pathogens. The research will not only help farmers but also the world economy, the climate and people in countries where food is scarce.

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Much of the world’s food is devastated every year when bacteria, fungi and viruses attack plants and kill them or suck all the nutrients and energy out of them.

Parasitic pathogens are the culprit, attacking crops such as wheat, barley, potatoes, apples and the grapes in vineyards on the slopes in Bourgogne.

Researchers around the world are striving to solve this problem, and Danish researchers are focusing on a whole new way of making plants more resistant to plant pathogens.

If the Danish researchers succeed with their strategy, it could greatly increase the yields of a wide range of crops. This can help not only farmers but also the world economy, the climate and people in countries that do not have the same easy access to food as in Denmark.

“Crop diseases is a major topic globally because of the high cost of yield losses. Farmers lose money and forests are cut to make space for new fields to compensate for the reduced yields. In addition, we use huge quantities of resources in the form of machines, fertilizer and pesticides to produce crops that end up failing anyway,” explains Hans Thordal-Christensen, Professor at Department of Plant and Environmental Sciences, University of Copenhagen.

In 2019, the Novo Nordisk Foundation awarded Hans Thordal-Christensen a Challenge Programme grant over 6 years for developing methods to make plants more resistant to pathogens.

Barley and wheat only resist powdery mildew and rust fungi for a few years

Two examples of crop-infecting pathogens are powdery mildew and rust fungi. Both types of fungi attack crops such as barley and wheat, and create major problems for farmers around the world, including Denmark.

Hans Thordal-Christensen says that there are currently regional epidemics of wheat stripe rust across the world.

The problem arises when wheat loses its resistance to the pathogens and then the farmers’ yields plummet.

Plant breeders are therefore constantly developing new varieties that can resist rust fungi.

This is an endless race against time, because every time plant breeders spend years and millions of dollars developing a new wheat variety that resists rust fungi, no more than 2–3 years elapse before the rust fungi find new ways to circumvent the wheat’s immune system.

“The plant breeders then need to start from scratch. Alternative ways are therefore needed for developing varieties of wheat with new forms of resistance that the rust fungi cannot circumvent as quickly as they can today,” explains Hans Thordal-Christensen.

Plant breeders spend millions on developing resistant plants

What do plant breeders currently do to develop new and more resistant varieties of crops?

The conventional way is to find wild strains of wheat and other crops that rust fungi have not yet conquered.

Such strains are found in the regions in which wheat originated, including Turkey, Iran, Syria and Israel, where wheat grows wild.

Wheat strains that resist rust fungi are present in these countries, but their yields are so pitiful that they are not commercially viable as crops.

The art of plant breeding therefore involves crossing the wild strains of wheat with the commercially promising varieties to create new varieties that have high yields and resist rust fungi.

“In the process, both undesirable and desirable characteristics can arise that affect such features as resistance to pathogens or drought and crop yield. The problem is that identifying the best new strains requires experimenting with thousands of new strains to find perhaps five strains that are of interest to farmers. This is really expensive, and the plant breeders have to repeat the whole process again when the rust fungi find new ways to circumvent the resistance,” says Hans Thordal-Christensen.

Fungi redirect nutrients

Understanding the new method of developing new and more resistant crop varieties requires understanding how pathogens such as fungi infect a plant and end up reducing the yield.

When a fungus lands on a plant, it transfers virulence proteins called effectors into the plant cells.

Plant pathogens have many of these effectors. Powdery mildew has about 800, and rust fungi have more than 1000.

Effectors manipulate the processes in the plant cells to benefit the pathogen instead of the plant.

This means that the effectors disarm the plant’s immune system. All plants have very effective defences against pathogens that enables them to resist infection.

However, if the pathogens can circumvent the immune system with their effectors, they can disable it while using the plant to obtain nutrients that enable the pathogen to grow.

“The fungus simply redirects the flow of nutrients so that it can absorb them instead of the plant. The powdery mildew and rust fungi we focus on also insert a specialized feeding structure called a haustorium into the plant cells. This is very important for the fungi’s ability to take control of the plant,” says Hans Thordal-Christensen, who also explains that the fungi use the haustoria to keep the plant cells alive while sucking nutrients and energy out of them, since these fungi cannot survive on dead cells.

“The question is how the fungi manipulate the plants to generate the membrane around the haustoria and how they use the haustoria to transport nutrients from the plant to the fungi. One third of the research project is focusing on elucidating this,” adds Hans Thordal-Christensen.

Inspired by bacteria that cause pneumonia

Hans Thordal-Christensen and his colleagues already have an idea about how to pry the secrets from the fungi.

The whole system of haustoria and membranes is similar to systems elsewhere in disease biology, including when Legionella bacteria infect humans and cause pneumonia.

Quite thorough research has shown how Legionella enters the cells of the lungs using effectors that take control of various membrane systems by forming a protective membrane around the bacterium.

Hans Thordal-Christensen speculates that powdery mildew and rust fungi do something similar.

“Achieving this requires an immensely complex system of many proteins, which is why the fungi have so many effectors. Researchers in human biology are much further ahead of us in this field. This can be annoying, but is also convenient, since they can inspire us,” explains Hans Thordal-Christensen.

A plant that totally resists powdery mildew and rust fungi

Hans Thordal-Christensen explains that when powdery mildew or rust fungi infect a plant using effectors to take control of the plant’s membrane proteins, then the plant is in the grip of the fungi.

But there is a little twist that underlies the entire research project:

Arabidopsis thaliana is the world’s most thoroughly studied plant. It is genetically similar to barley and wheat, although these plants look very different. However, if researchers try to infect Arabidopsis thaliana with cereal powdery mildew or rust fungi, nothing happens.

Despite their otherwise extremely well-developed arsenal of weapons to penetrate plant cells and take control of them, these fungi cannot infect Arabidopsis thaliana. The plant is 100% resistant and will remain so in the foreseeable future.

Arabidopsis thaliana activates its defences, suppresses the fungi and does not get sick. It defends itself and is not susceptible because the effectors are not compatible with the proteins in Arabidopsis thaliana,” explains Hans Thordal-Christensen.

Creating barley and wheat with the same immune systems as Arabidopsis thaliana

Hans Thordal-Christensen’s research project will attempt to develop the immune systems of barley and wheat by copying from Arabidopsis thaliana.

The researchers will identify the effectors in powdery mildew and rust fungi that contribute most to infecting barley and wheat. Then they will identify the proteins that the effectors target inside the plants. Finally, the researchers will change the effectors’ target proteins in barley and wheat plants to resemble the Arabidopsis thaliana proteins, which the effectors cannot manipulate.

The desired result is that the researchers could infect the barley and wheat plants with powdery mildew and rust fungi – and nothing would happen.

“We probably cannot achieve this by making just one change, because the fungi will probably be able to figure out how to circumvent that part of the immune system. We need to introduce several changes to multiple proteins: five or six. This will make the plants resist the fungi based on several different types of defences that the fungi must circumvent. They cannot do this because that means they would have to develop new effectors against all the altered proteins at the same time,” says Hans Thordal-Christensen.

Identified the first 100 effectors in powdery mildew

Although the researchers behind the project have only taken the initial steps towards developing new and more resistant crops, the research is based on 30 years of work to understand how plants defend themselves against fungi and how fungi attack plants.

Hans Thordal-Christensen and his colleagues were at the forefront of studying the effectors in powdery mildew that culminated in a project in 2010, when they compared their own gene sequence data from the powdery mildew with large genetic databases and found that these 100 proteins are unique and can therefore be suspected of being effectors.

“We found about 100 unknown proteins with this signal peptide. Further, we could not find them in other species of fungi except in the powdery mildew fungi. Normally, when we find a protein in one fungus, we usually find a similar protein in almost all other eukaryotic cells but not in this case. We knew from other people’s experiments with potato blight that effectors were very unique to the individual pathogen. This led us to conclude that these 100 proteins are effectors,” explains Hans Thordal-Christensen.

Further studies on the effectors are still underway, and so far, the researchers know very little about what their functions are during infection.

Gene mutation enables powdery mildew to attack Arabidopsis thaliana

Another important project in the run-up to the new research is a discovery that changed Hans Thordal-Christensen’s life.

More than 20 years ago, he conducted research on Arabidopsis thaliana in a laboratory in the United States.

There he discovered a mutation in the PEN1 gene, whose function was not known at the time.

Hans Thordal-Christensen’s research showed, however, that PEN1 is a key actor in a plant’s defence to prevent fungi from penetrating the plant’s cells.

More specifically, PEN1 is involved in membrane traffic, and this is where Hans Thordal-Christensen’s interest in membranes began.

The revealing aspect of the research was that when PEN1 was mutated, the otherwise solid defences of Arabidopsis thaliana against powdery mildew disappeared, and a fungus that normally could not infect Arabidopsis thaliana suddenly ran riot.

“The mapping of the function of PEN1 is a milestone that changed my life, and research on PEN1 and membranes has really interested me since then. An intact immune system is required to prevent the powdery mildew from infecting Arabidopsis thaliana, and if we disable the immune system by mutating PEN1, we help the fungus to infect a plant, which it would otherwise not be able to,” says Hans Thordal-Christensen.

Discovery reveals how the membrane may be formed

The third research project that brought the researchers to where they are today focused on the membranes that surround the haustoria.

In 2017, Hans Thordal-Christensen’s group discovered that the membrane, which the powdery mildew fungus stimulates the plant cells to generate around haustoria, shares properties with the endoplasmic reticulum membrane.

Further, the researchers excluded various possible explanations for how the membranes around the haustoria are formed.

This left them with one possibility that they will examine in the new research project.

“We suggest that the membranes are made by lipid transfer proteins that can move lipids from one membrane to another. They work very fast, and we now believe that the effectors take control of these lipid transfer proteins and that is how the membranes around the haustoria are formed,” explains Hans Thordal-Christensen.

What the researchers would like to achieve in 6 years

Looking 6 years ahead, Hans Thordal-Christensen hopes that the overall research will result in the researchers identifying five or six potential proteins in barley and wheat that they can manipulate to resist the fungal effectors.

This will open up opportunities for creating superresistant strains of barley and wheat, which will lead to higher crop yields.

But Hans Thordal-Christensen also hopes that the research from this major project will produce substantial knowledge that can be valuable in other ways in the struggle for the world’s crops and in understanding plants’ immune systems and the struggle against pathogens.

“If we get there, I will be very satisfied. If we get further, we may have implemented some of these proteins in barley or wheat and will be in the process of testing them. This will be absolutely amazing,” says Hans Thordal-Christensen.

In 2019, the Novo Nordisk Foundation awarded a Challenge Programme grant to Hans Thordal-Christensen for the project “A new paradigm for disease-free crops of tomorrow”.

Plant diseases are highly problematic in crop production, and pathogen control using agrochemicals is costly and environmentally damaging. For many ye...

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