Many of the bacteria that compromise crops and jeopardize our food sources deploy a common strategy to cause disease: they release a blend of detrimental proteins straight into the plant cells.
For over two decades, a team of researchers from Duke University has been studying this set of molecules that plant pathogens use to cause diseases in a variety of crops worldwide, ranging from rice to apple trees.
Now, a collective initiative from three collaborating research teams may have found the answer to how these molecules induce illness in plants, while providing a possible method to neutralize them.
The researchers examined key elements of this deadly blend, focusing on a group of injected proteins named AvrE/DspE. These proteins are accountable for diseases like brown spot in beans, bacterial speck in tomatoes, and fire blight in fruit trees.
Since its discovery in the early 1990s, this protein group has grabbed the attention of many experts in plant disease. Because these proteins are a vital tool for bacteria, deactivating them in controlled settings renders the bacteria harmless.
Yet, despite decades of research, the exact mechanisms through which they infect plant cells have remained elusive.
Now, the scientists managed to identify several proteins in the AvrE/DspE group that suppressed the plants’ defense mechanisms, often leading to distinct, waterlogged spots on their leaves, which is a telltale sign of disease.
While the sequence of amino acids that linked to form the proteins was already understood, the three-dimensional folding of these sequences remained a mystery.
A notable challenge was the sheer size of the proteins: while a typical bacterial protein might contain around 300 amino acids, the proteins from the AvrE/DspE family boast a staggering 2000. Moreover, efforts to find proteins with similar sequences proved fruitless. “They’re weird proteins,” said senior author Sheng Yang He, a biologist at Duke.
To address this problem, the researchers used a 2021 computer program named AlphaFold2, which employs artificial intelligence to predict the 3D formation of a particular amino acid sequence.
While the experts already knew that some members of this family help the bacteria evade the plants’ immune systems, a first look into the proteins’ 3D design suggested they have an additional role.
“When we first saw the model, it was nothing like what we had thought,” confessed Pei Zhou, a professor of Biochemistry at Duke who collaborated with the study authors.
AI predictions for bacterial proteins impacting crops like pears, apples, tomatoes, and corn, all hinted at a consistent 3D design, resembling a tiny mushroom with a tubular stem. This predicted shape matched up with images of a protein responsible for fire blight disease in fruit trees, which were captured via a cryo-electron microscope.
Seen from above, the protein was highly similar to an empty hollow tube. This led researchers to an intriguing hypothesis: perhaps bacteria employ these proteins to puncture the plant cell membrane, or, as He argued, to “force the host for a drink” during infection.
Once they enter the leaves, the bacteria first encounter an intercellular area called the apoplast. Plants typically maintain this space in a dry state, facilitating gas exchanges that are crucial for photosynthesis. However, during bacterial invasion, the inside of the leaves becomes waterlogged, making it ripe for bacterial sustenance and proliferation.
Further probing into the predicted 3D model for the fire blight-causing protein revealed that while the exterior of this tubular form repelled water, its inner core had an increased affinity for water.
To test this “water channel hypothesis,” the researchers collaborated with Duke’s biology experts Ke Dong and Felipe Andreazza, who added the gene readouts for the bacterial proteins AvrE and DspE to frog eggs, converting these eggs into protein factories. Once introduced into a saline environment, the eggs expanded due to excessive water and subsequently ruptured.
In their quest to neutralize the damaging proteins by blocking their channels, the experts focused on a type of minute spherical nanoparticles known as PAMAM dendrimers. These particles have been used for decades in drug delivery and can be designed with precision in the lab.
“We were tinkering with the hypothesis that if we found the right diameter chemical, maybe we could block the pore,” He explained.
After testing different sized particles, they identified one with the right size to obstruct the water channel protein from the fire blight bacterium, Erwinia amylovora.
By taking frog eggs designed to synthetize this protein and dousing them with the PAMAM nanoparticles, they managed to successfully stop the water flowing into the eggs.
Similar tests on Arabidopsis plants afflicted with the Pseudomonas syringae bacterium – a known cause of bacterial speck – confirmed the nanoparticles’ efficacy. These particles successfully stopped the bacteria from taking hold, reducing its concentration in plant leaves by a hundredfold.
Tests were extended to pear fruits exposed to fire blight causing bacteria. Remarkably, the fruits remained healthy and symptom-free. “It was a long shot, but it worked,” He said. “We’re excited about this.”
According to the experts, these findings could prove revolutionary in combating numerous plant diseases. While plants contribute to 80 percent of our diet, over 10 percent of global crop yield, including crops such as wheat, rice, maize, potato, and soybean, succumb to pests and pathogens each year, leading to a staggering economic loss of $220 billion.
In future research, the scientists plan to clarify how this protection works by examining more closely how the channel-blocking nanoparticles and the channel proteins interact.
“If we can image those structures, we can have a better understanding and come up with better designs for crop protection,” Zhou concluded.
The study is published in the journal Nature.
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