Duke University researchers may have discovered a method to neutralize them, potentially averting $220 billion in annual agricultural losses.
Many of the bacteria that ravage crops and threaten our food supply use a common tactic to cause disease: they inject a cocktail of harmful proteins directly into the plant’s cells.
For 25 years, biologist Sheng-Yang He and his senior research associate Kinya Nomura have been investigating this set of molecules that plant pathogens use to cause disease in hundreds of crops worldwide, from rice to apple orchards.
Now, thanks to a team effort by three collaborating research groups, they may finally have an answer to how these molecules cause plant disease—and a way to disarm them.
The findings appear in the Sept. 13 issue of the journal Nature.
Researchers in He’s lab are studying key components of this deadly cocktail, a family of injected proteins called AvrE/DspE that cause diseases ranging from bean brown spot and tomato bacterial spot to fruit tree blight.
Ever since their discovery in the early 1990s, this family of proteins has been of great interest to those studying plant diseases. They are key weapons in the bacterial arsenal; knocking them out in the lab renders otherwise dangerous bacteria harmless. But despite decades of effort, many questions about how they work remain unanswered.
The researchers identified a number of proteins in the AvrE/DspE family that suppressed the plant’s immune system or that caused dark water-soaked spots on the plant’s leaves — the first signs of infection. They even knew the basic sequence amino acids which joined together to form proteins, like beads on a string. But they didn’t know how this chain of amino acids folded into a 3D shape, so they couldn’t easily explain how they worked.
Part of the problem is that the proteins in this family are huge. With regard to it regarding to it, average bacterial protein can be 300 amino acids in length; The AvrE/DspE-family of proteins is 2,000.
The researchers looked for other proteins with similar sequences for clues, but none with known functions turned up.
“They’re weird proteins,” he said.
So they turned to a computer program released in 2021 called AlphaFold2, which uses artificial intelligence to predict what 3D shape a given chain of amino acids will have.
Scientists knew that some members of this family help bacteria evade the plant’s immune system. But their first look at the 3D structure of the proteins suggested another role.
“When we first saw the model, it was nothing like what we thought,” said study co-author Pei Zhou, a professor of biochemistry at Duke whose lab contributed to the findings.
The researchers looked at AI predictions for bacterial proteins that infect crops including pears, apples, tomatoes and corn, and all pointed to a similar 3D structure. They seemed to fold into a small mushroom with a cylindrical straw-like stem.
The predicted shape matched well with cryo-electron microscope images of the bacterial protein that causes blight in fruit trees. From top to bottom, this protein looked very much like a hollow tube.
That got the scientists thinking: Maybe the bacteria use these proteins to punch a hole in the plant cell membrane to “force the host to drink” during infection, he said.
Once bacteria enter leaves, one of the first areas they encounter is the space between cells called the apoplast. Usually plants keep this area dry to allow gas exchange photosynthesis. However, when the bacteria invade, the inside of the leaf becomes waterlogged, creating a moist cozy haven for them to feed and multiply.
Further examination of the predicted 3D model for the fire blight protein revealed that while the exterior of the straw-like structure is resistant to water, its hollow inner core has a special affinity for water.
To test the water channel hypothesis, the team joined forces with biology professor Duke Ke Dong and co-author Felipe Andreazza, a postdoctoral fellow in her lab. They added the gene data for the bacterial proteins AvrE and DspE to frog eggs, using the eggs as cellular factories to make the proteins. Eggs placed in dilute saline quickly swelled and burst with too much water.
The researchers also tried to see if they could disarm these bacterial proteins by blocking their channels. Nomura focused on a class of small spherical nanoparticles called PAMAM dendrimers. These dendrimers, which have been used for more than two decades in drug delivery, can be made with precise diameters in the laboratory.
“We were working with the hypothesis that if we could find a chemical with the right diameter, maybe we could block the pore,” he said.
After testing different sized particles, they identified one they thought might be just the right size to disrupt a water channel protein produced by the downy mildew pathogen, Erwinia amylovora.
They took frog eggs engineered to synthesize this protein and doused them with PAMAM nanoparticles, and water stopped flowing into the eggs. They didn’t flow.
They also treated Arabidopsis plants infected with the bacterial spot pathogen Pseudomonas syringae. The channel-blocking nanoparticles prevented bacteria from taking hold and reduced pathogen concentrations in plant leaves a hundredfold.
The compounds were also effective against other bacterial infections. The researchers did the same with pear fruit exposed to the bacteria that causes the fungal disease, and the fruit never developed symptoms—the bacteria did not cause the disease.
“It was a long shot, but it worked,” he said. “We’re excited about it.
The findings could offer a new line of attack against many plant diseases, the researchers said.
Plants produce 80% of the food we are eating. And even more than 10% Global food production—crops such as wheat, rice, corn, potatoes, and soybeans—is lost each year to plant pathogens and pests, costing the global economy enormous 220 billion dollars.
The team has filed a provisional patent on the approach.
The next step, said Zhou and co-author Jie Cheng, Ph.D. student in Zhou’s lab, is to figure out how this protection works by looking more closely at how channel-blocking nanoparticles and channel proteins interact.
“If we can image these structures, we can better understand them and come up with better designs for crop protection,” Zhou said.
Reference: “Bacterial pathogens provide water- and solute-permeable channels to plant cells” by Kinya Nomura, Felipe Andreazza, Jie Cheng, Ke Dong, Pei Zhou, and Sheng Yang He, 13 Sep 2023, Nature.
The study was funded by the National Institute of Allergy and Infectious Diseases and the National Institute of General Medical Sciences, both at National Institute of HealthDuke Science and Technology and the Howard Hughes Medical Institute.