Duke University researchers may have found a way to neutralize them, potentially preventing $220 billion in annual agricultural losses.
Many bacteria that destroy crops and threaten our food supply use a common strategy to cause disease: They inject a cocktail of harmful proteins directly into plant cells. For 25 years, biologist Shengyang He and his senior researcher Kinya Nomura have studied a group of molecules used by plant pathogens to cause disease in hundreds of crops around the world, from rice to apple orchards. The results were published on September 13 in the journal Nature. Researchers in He’s lab studied the key ingredient in this deadly cocktail, a family of injectable proteins called AvrE/DspE, which cause diseases ranging from brown spot on beans to bacterial spot on tomatoes to fruit trees. In the early 1990s, this protein family attracted great interest among plant disease researchers. They are the main weapons in bacterial arsenals; their elimination in the laboratory renders dangerous bacteria harmless. But despite decades of effort, many questions about how it works remain unanswered.
Researchers have identified several proteins in the AvrE/DspE family that can suppress a plant’s immune system or cause black, water-soaked spots on plant leaves, the first sign of infection. They even know the basic sequence of amino acids that together make proteins, like beads on a string. But they didn’t know how a string of amino acids folded into a 3D shape, so they couldn’t easily explain how they worked. Part of the problem is the large number of proteins in this family. The average length of bacterial proteins can be 300 amino acids; For proteins of the AvrE/DspE family, it is 2,000. The researchers have looked for other proteins with similar sequences for clues, but have not found any with a known function.
They are strange proteins,” he said. So they turned to a computer program released in 2021 called AlphaFold2, which uses artificial intelligence to predict the 3D shape a given sequence of amino acids will take.
Scientists know that some members of this family help bacteria evade the plant’s immune system. But for the first time, they have seen the 3D structures of these proteins, suggesting an additional role. When we first saw this model, it was nothing like we imagined. Pei Zhou, co-author of the study and professor of biochemistry at Duke University, said his lab produced the results.
The researchers studied AI predictions of bacterial proteins that infect crops such as pears, apples, tomatoes and corn, and all the results pointed to similar 3D structures. They appear to fold into a small mushroom with a cylindrical stem like a straw.
The predicted shape closely matched images of the bacterial protein that causes fruit tree fire, taken using cryo-electron microscopy. When viewed from the top down, the protein looks like a hollow tube.
That led the researchers to speculate: Perhaps the bacteria use these proteins to punch holes in plant cell membranes, “forcing the host to drink water” during infection, he said. When bacteria enter a leaf, one of the first places they encounter is the space between cells called the apoplast. Normally, plants keep the area dry to allow the exchange of photosynthetic gases. But when the bacteria enter, the inside of the leaf becomes saturated with water, creating a moist and comfortable haven for the bacteria to feed and multiply.
Further examination of the 3D model of the predicted fire-prone protein showed that while the exterior of the straw-like structure is impermeable, its hollow core has a special affinity for water. To test the water channel hypothesis, the team collaborated with Duke biology professor Ke Dong and co-author Felipe Andreazza, a postdoctoral fellow in his lab. They added the genetic readings for the bacterial proteins AvrE and DspE into frog eggs, using the eggs as cellular factories to produce the proteins. Eggs placed in a dilute salt solution will quickly swell and burst from the excess water.
Researchers are also trying to see if they can disarm these bacterial proteins by blocking their pathways. Nomura focuses on a class of small spherical nanoparticles called PAMAM dendrimers. These dendrimers, which have been used for drug delivery for more than two decades, can be produced in the laboratory with precise diameters. “We’re modifying the hypothesis that if we find chemicals with the right diameter, we could block the pore,” he said. After testing different particle sizes, they found a size they thought might be suitable for clogging waterways. A protein produced by the fire rot pathogen E. amylovora.
They took frog eggs that had been modified to synthesize the protein and impregnated the eggs with PAMAM nanoparticles, which stopped water from entering the eggs. They do not swell.
They also treated Arabidopsis plants infected with the pathogen Pseudomonas syringae, which causes bacterial spot. The channel-blocking nanoparticles stopped bacterial growth, reducing pathogen concentrations in plant leaves by 100-fold.
These compounds are also effective against other bacterial infections. The researchers did the same with stone fruits exposed to fire blight bacteria, and the fruit never developed symptoms—the bacteria didn’t make them sick.
“It’s a long shot, but it’s working,” he said. “We’re excited about it.”
Researchers say the findings could lead to new treatments for many plant diseases.
80% of the food we eat is produced by plants. However, each year more than 10% of global food production (such as wheat, rice, corn, potatoes and soybeans) is lost to plant pathogens and pests, costing the global economy up to $220 billion. The team has filed a provisional patent application for the method.
The next step, said Zhou and first author Jie Cheng, a Ph.D. Students in Zhou’s lab will learn how this protection works by taking a closer look at the interactions between channel-blocking nanoparticles and channel proteins. “If we can image these structures, we can better understand and come up with better crop protection models,” Zhou said.
Citation: “Bacterial pathogens provide water and solute channels permeable to plant cells,” Kinya Nomura, Felipe Andreazza, Jie Cheng, Ke Dong, Pei Zhou, and Shen Yang He, 13 September 2023, Nature.